JP2023537237A - Crystalline salts of tizoxanide and 2-hydroxy-N-(5-chloro-1,3-thiazol-2-yl)benzamide (RM-4848) with ethanolamine, morpholine, propanolamine, piperazine, and N-methylpiperazine - Google Patents

Abstract

The present invention provides crystallized amine salts of tizoxanide, such as ethanolamine salts, morpholine salts, propanolamine salts, piperazine salts, and N-methylpiperazine salts, such as 2-hydroxy-N-(5-halo-1, Crystallized amine salts of 3-thiazol-2-yl)benzamide derivatives (such as 2-hydroxy-N-(5-chloro-1,3-thiazol-2-yl)benzamide (RM-4848)), thizo Methods for preparing xanidoamine salts from their prodrug nitazoxanide (NTZ), pharmaceutical compositions containing tizoxanide amine salts, tizoxanide amine salts for use as antiviral or antiparasitic agents Regarding.

Description

RELATED APPLICATIONS This application claims priority to United States Provisional Patent Application No. 63/054,072, filed July 20, 2020, which is hereby incorporated by reference in its entirety.

The present disclosure relates to thiazolide compounds, and more particularly salts of thiazolide compounds, and methods of making and using the same.

One embodiment has the formula:
is an amine containing salt of a compound with where R is _NO2 or a halogen.

Another embodiment is a pharmaceutical composition comprising a salt of tizoxanide and a pharmaceutically acceptable excipient, wherein the composition, when administered to a mammal, has a maximum concentration of tizoxanide in the plasma of the mammal of provided in less than an hour.

Yet another embodiment is a pharmaceutical composition comprising a salt of tizoxanide and a pharmaceutically acceptable excipient, wherein the composition, when administered to a mammal, comprises of the compound in plasma more quickly than pharmaceutical compositions containing nitazoxanide.

Yet another embodiment is a pharmaceutical composition comprising a salt of tizoxanide and a pharmaceutically acceptable excipient, wherein the composition, when administered to a mammal, comprises in the plasma of tizoxanide _and glucoronotizoxanide at or above the AUC _0-12h concentrations of pharmaceutical compositions comprising nitazoxanide.

Yet another embodiment is a method of making an amine-containing salt of a thiazolide compound, the method comprising the formula:
with _an amine-containing compound to form an amine-containing salt of the thiazolide compound.

Yet another embodiment is the ethanolamine of tizoxanide.

FIG. 1 is a plot showing the median plasma tizoxanide (T) concentration (μg/mL) over 12 hours for RM-5071, RM-5072, and nitazoxanide (NTZ). FIG. 2 is a plot showing the median plasma tizoxanide glucuronide (TG) concentration (μg/mL) over 12 hours for RM-5071m, RM-5072, and NTZ. FIG. 3 is a plot showing the median plasma free and glucuronidated tizoxanide concentrations (μg/mL) for RM-5071, RM-5072, and nitazoxanide (NTZ) over 12 hours. FIG. 4 shows examples of thiazolidamine-containing salts. Figure 5 shows potential impurities within a batch of RM-5071. Figure 6 shows scanning electron microscopy (SEM) images of a batch of RM-5071 at magnifications of 500x (top) and 1000x (bottom). Figure 7 shows SEM images of a batch of desacetyl NTZ (tizoxanide) at magnifications of 500x (top) and 1000x (bottom). FIG. 8 shows digital images (10× magnification) of primary particles before (left) and after (right) sonication of a sample preparation for particle analysis. Figure 9 shows particle measurements at different applied pressures of 0 bar (green), 1 bar (blue), 2 bar (purple = grey-blue) and 4 bar (grey) overlaid with the average of the liquid dispersion (red). indicates Pressures 3 and 4 overlap. Figure 10 shows a liquid analysis (red) and an overlay of 4 bar (green) and 1 bar (blue) pre-dispersion and high energy venture analyses. Figures 11A-B show thermogravimetric analysis (TGA) thermograms of RM-5071 (A) and desacetyl-NTZA (B). Figures 12A-B show differential scanning calorimetry (DSC) thermograms of RM-5071 (A) and desacetyl-NTZA (B). Figure 13 shows the titration curve of a 1 mg/mL solution of RM-5071 with 0.01N HCl. Figure 14 shows the titration curve of a 1 mg/mL solution of RM-5071 with 0.02N NaOH. FIG. 15 represents the UV-Vis absorption spectrum of RM-5071 dissolved in methanol. Figure 16 is the absorbance of RM-5071 (λmax = 409 nm) as a function of solution concentration in water-supernatant. Figure 17 is the H-NMR spectrum of RM-5071, obtained by the NMR method ( ^1H ) using a 400 MHz instrument. FIG. 18 is the 1 H-NMR spectrum corresponding to the region of the spectrum of the aromatic functional groups of RM-5071. Figure 19 is the H-NMR spectrum corresponding to the aliphatic protons of RM-5071. FIG. 20 shows positive (top) and negative (bottom) electrospray ionization mass spectrometry (ESI-MS) spectra for RM-5071. FIG. 21 shows another positive (top-blue) and negative (bottom-black) electrospray ionization mass spectrometry (ESI-MS) spectra for RM-5071. Figure 22 is an overlay of the Fourier transform infrared (FTIR) spectra of RM-5071 (red) and desacetyl-NTZA (blue). FIG. 23 schematically shows the resonance structure for RM-5071. Figure 24 shows the attenuated total reflectance Fourier transform infrared (FTIR) spectra of RM-5071 (red), desacetyl-NTZA (green), and a 1:1 mixture of desacetyl-NTZA and ethanolamine (black). Figures 25A-B show the X-ray diffractograms of RM-5071 (A) and desacetyl-NTZA (B). Figures 25A-B show the X-ray diffractograms of RM-5071 (A) and desacetyl-NTZA (B).

In this specification and claims, the singular forms "a," "one," and "the" include plural unless the context clearly dictates otherwise. Throughout this specification, unless otherwise stated, the terms "including" and "including" are inclusive rather than exclusive, such that a stated integer or group of integers may refer to one or more other stated It can contain integers or groups of integers that are not The term "or" is inclusive unless modified, for example, by "either." Thus, unless the context indicates otherwise, the term "or" means any one member of a specific list and includes any combination of members of that list. Except in the examples or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions utilized herein are modified at all times by the term "about." should be understood.

Headings are provided for convenience only and are not to be construed as limiting the invention in any way. Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the claims. To make this disclosure easier to understand, some terms are first defined.

All numerical designations (eg, pH, temperature, time, concentration, and molecular weight) are approximate and vary (+) or (-) by 1, 5, or 10% increments. Although not always explicit, it should be understood that the term "about" precedes any numerical value specified. Although not always explicitly stated, the reagents described herein are merely exemplary and equivalents known in the art are indicated throughout the detailed description. It should also be understood that

"NMR" means nuclear magnetic resonance.

"Veq" means the volume of EQuivalence points.

" _AUC0-12h " means the total area below plasma concentration from time zero (ie, the time of administration) to 12 hours after administration.

_Cmax means the maximum plasma or serum concentration reached by a drug after administration.

"FTIR" means Fourier transform infrared spectroscopy.

"UV" means ultraviolet and visible light spectroscopy.

"DMF" means dimethylformamide.

"DMA" means dimethylacetamide.

"PO" means oral.

NTZ or NTZA means nitazoxanide (also known as 2-(acetryloxy)-N-(5-nitro-2-thiazolyl)benzamide) and has the following structure:
is a compound with

TIZ, desacetyl-NTZA, desacetyl-NTZ, or desacetylnitazoxanide, is the circulating active metabolite of nitazoxanide. Tizoxanide has the formula:
have.

Another metabolite of nitazoxanide is glucoronotizoxanide, which has the formula:
have.

Nitazoxanide is approved in the United States for the treatment of diarrhea caused by Cryptosporidium parvum and Giardia lamblia.

RM-4848 is a substituted thiazolide, with the same structure as tizoxanide, but with a chloro group instead of a nitro group, resulting in the compound N-(5-chlorothiazol-2-yl)-2-hydroxybenzamide. RM-4848 has the following formula:
have.

Thiazolide compounds can be synthesized, for example, according to procedures published in US Pat.

Pharmaceutical compositions containing nitazoxanide and its metabolite tizoxanide were originally developed and marketed to treat intestinal parasitic infections. Various applications of nitazoxanide, tizoxanide, and other thiazolide compounds (such as RM-4848) are described, for example, in U.S. Pat. 10,100,023, RE46,724, 9,827,227, 9,820,975, 9,351,937, 9,345,690, 9,126,992, 9,107,913, 9,023,877, 8,895,752, No. 8,846,727, No. 8,772,502 8,633,230, 8,524,278, 8,124,632, 7,645,783, 7,550,493, 7,285,567, 6,117,894, 6,020,353, 5,968,961, 5,965,590 , No. 5,935,591, No. 5,886,013, 5,859,038, 5,856,348, and U.S. Patent Application Publication Nos. 20200038377, 20190321338, 20190307730, 20190291404, 20190276417, 20190040026, 2018012 6722, 20180085353, 2018078533, No. 20170334868, No. 20170281603, No. 20160243087, No. 20160228415, No. 2015025768, No. 20140341850, No. 20140112888, No. 20140065215, No. 2012029483 No. 1, No. 20120122939, No. 20120108592, No. 20120108591, No. 20100330173 20100292274, 20100209505, 20090036467, 20080097106, 20080097106, 20080096941, 20070167504, 20070015803, 200601 94853, 20060089396, 20050171169 and , each of which is incorporated herein by reference in its entirety.

We have the following formula:
developed novel salts of thiazolide compounds of where R is _NO2 or halogen (such as Cl or Br).

In some embodiments, the salt of the thiazolide compound can be an amine-containing salt. As used herein, the term amine-containing salt means a salt that has a counterion and contains one or more amine groups, such as primary, secondary, or tertiary amine groups. do.

In some embodiments, the amine-containing salt can be an alkylamine, oxaalkylamine, or cycloalkylamine salt.

As used herein, an "alkylamine" can be an alkyl group having one or more amine groups, such as primary, secondary, or tertiary amine groups.

As used herein, the term "alkyl", alone or in combination, refers to a straight or branched chain containing 1 to 10, 1 to 6, or 1 to 4 carbon atoms. means an alkyl group of The term "alkyl group" can be used in its broadest sense. Alkyl groups may be optionally substituted. Examples of alkyl groups include ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, pentyl, iso-amyl, hexyl.

In some embodiments, an alkylamine can be an alkyl containing one or more terminal amino groups. Examples of such alkylamines include ethylamine, n-propylamine, n-butylamine, s-butylamine, t-butylamine, and isobutylamine.

In some embodiments, an alkylamine can be an alkyl having one or more _CH2 groups substituted with NH.

As used herein, oxaalkyl means an alkyl having one or more _CH2 and/or _CH3 groups substituted with O and/or OH.

In some embodiments, an oxaalkylamine can be an alkyl with a terminal OH group and a terminal amino group. Examples of such amines include ethanolamine, propanolamine, n-butanolamine.

The term "cycloalkyl," as used herein, alone or in combination, contains 3-12, 3-8, or 3-6 carbon atom ring members in each ring moiety, which is It means an optionally substituted saturated monocyclic group. Examples of such cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, octahydronaphthyl, 2,3-dihydro-lH-indenyl, adamantyl, and the like.

Cycloalkylamine means a cycloalkyl with one or more _CH2 groups substituted with NH. In some embodiments, the cycloalkylamine can be a cycloalkyl with one _CH2 group substituted with NH. Furthermore, in some embodiments, the cycloalkylamine can be a cycloalkyl having more than one, ie two or more _CH2 groups substituted with NH.

In some embodiments, one or more _CH2 groups in the cycloalkylamine may be further substituted with O or _CH3N .

Examples of cycloalkylamines include morpholine and N-methylpiperazine.

In some embodiments, the amine-containing salt can be an ethanolamine salt, a morpholine salt, a propanolamine salt, or an N-methylpiperazine salt.

In some embodiments, the amine-containing salt of a thiazolide compound can be a salt of a liquid amine containing a base, where the base is ammonia, methylamine, diethylamine, ethanolamine, dicyclohexylamine, N-methylmorpholine, ammonium , tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, di benzylamine, N,N-dibenzylphenethylamine, 1-ephenamine, and N,N'-dibenzylethylenediamine, ethylenediamine, ethanolamine, diethanolamine, piperidine and the like.

In some embodiments, the amine-containing salt can be a crystalline salt

In some embodiments, at least 90%, or at least 92%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 98.5% as amine-containing salts , or at least 99%, or at least 99.1%, or at least 99.2%, or at least 99.3%, or at least 99.4%, or at least 99.5% purity. The pure amine-containing salt contains at least 10 g, or at least 20 g, or at least 30 g, or at least 40 g, or at least 50 g, or at least 60 g, or at least 70 g, or at least 80 g, or at least 90 g. g, or at least 100 g, or at least 150 g, or at least 200 g, or at least 250 g, or at least 300 g, or at least 350 g, or at least 400 g, or at least 450 g, or at least 500 g, or at least 600 g , or at least 700 g, or at least 800 g, or at least 900 g, or at least 1000 g, or at least 1200 g, or at least 1400 g, or at least 1500 g salt, or at least 2000 g salt, or at least 4000 g salt, or at least 5000 g salt, or at least 8000 g salt, or at least 10000 g salt, or at least 15000 g salt, or at least 20000 g salt, or at least 25000 g salt, or at least 30000 g salt , or in batches containing at least 35000 g of salt, or at least 40000 g of salt.

In some embodiments, a salt of a thiazolide compound (such as a salt of an amine-containing thiazolide compound) can be administered as part of a pharmaceutical composition. The pharmaceutical composition can contain a carrier (such as a pharmaceutically acceptable carrier) in addition to the salt of the thiazolide compound. The term "substrate" can be used in its broadest sense. For example, the term "base" includes any base, diluent, excipient, wetting agent, buffer, suspending agent, lubricant, adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent, preserving agent. agents, surfactants, coloring agents, flavoring agents, and sweetening agents. In some embodiments, the carrier can be a pharmaceutically acceptable carrier, which is a narrower term than carrier. This is because the term "pharmaceutically acceptable carrier" means a non-toxic substance considered suitable for use in pharmaceutical compositions. The actual dosage level of an active ingredient in a pharmaceutical composition can vary so as to administer an effective amount of the active compound to achieve the desired therapeutic response for a particular patient.

The selected dose level may depend on the activity of the thiazolide compound, the route of administration, the severity of the condition being treated, and the condition and prior medical history of the patient being treated. However, it is within the skill in the art to start the dose of the compound at a level lower than that required to achieve the desired therapeutic effect and increase gradually until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for administration, eg, from 2 to 4 times daily. However, the specific dosage level for any particular patient will depend on a variety of factors, including body weight, general health, diet, time and route of administration and combination of other therapeutic agents, and the condition or disease being treated. severity included).

A pharmaceutical composition can be administered systemically, for example, in an oral formulation (such as a solid oral formulation). It can be in physical form such as powders, tablets, capsules, lozenges, gels, solutions, suspensions, syrups and the like. In some embodiments, pharmaceutical compositions can be in the form of formulations disclosed in US Pat. Nos. 8,524,278 and 9,351,937. Such formulations include, for example, a controlled release portion and an immediate release portion, such that at least one of the controlled release portion and the immediate release portion comprises a salt of a thiazolide compound (such an amine-containing salt of a thiazolide compound). be able to. For example, in some embodiments, the controlled-release portion can comprise a salt of a thiazolide compound (such as an amine-containing salt of a thiazolide compound), whereas the immediate-release portion can comprise a salt of a thiazolide compound (such as a salt of a controlled-release portion). salts therein, which may be the same or different), and/or the thiazolide compound itself. Further, in some embodiments, the immediate release moiety can comprise a salt of a thiazolide compound (such as an amine-containing salt of a thiazolide compound), whereas the controlled release moiety can comprise a salt of a thiazolide compound (such as an immediate release moiety). salts therein, which may be the same or different), and/or the thiazolide compound itself. These compositions can be administered in a single dose or in multiple doses administered at different times.

In some embodiments, the total amount of thiazolide compound in a composition containing a salt of a thiazolide compound (such as an amine-containing salt of a thiazolide compound) is from about 20% to about 95%, or about 30% by weight of the composition. % to about 90%, or about 35% to about 85%, or about 60% to about 75%. The composition can be formulated for immediate release, controlled release, or sustained release. The composition can contain one or more additional additives or excipients that are pharmaceutically acceptable. These excipients are therapeutically inactive ingredients that are well known and appreciated in the art. As used herein, the term "inert ingredient" can refer to therapeutically inactive ingredients well known in the art of pharmaceutical manufacturing, which can be used alone or in various combinations. , which include, for example, diluents, disintegrants, binders, suspending agents, glidants, lubricants, fillers, coatings, solubilizers, sweeteners, colorants, flavorants, and antioxidants. is an agent. See, eg, Remington: The Science and Practice of Pharmacy 1995, E. W. Martin, ed., Mack Publishing Company, 19th ed., Easton, Pennsylvania.

Non-limiting examples of diluents or fillers include starch, lactose, xylitol, sorbitol, powdered sugar for icing, compressed sugar, dextrates, dextrin, dextrose, fructose, lactitol, mannitol, sucrose, talc, Microcrystalline cellulose, calcium carbonate, dicalcium phosphate or tricalcium phosphate, dicalcium phosphate dehydrate, calcium sulfate and the like. The amount of diluent or filler can range from about 2% to about 15% by weight of the total composition.

Non-limiting examples of disintegrants include alginic acid, DVB methacrylate, cross-linked PVP, microcrystalline cellulose, croscarmellose sodium, crospovidone, potassium polacrilin, sodium starch glycolate, starch (cornstarch or corn starch, pre-gelatinized starch, etc.). Disintegrants typically comprise from about 2% to about 15% by weight of the total composition.

Non-limiting examples of binders include starches (potato starch, wheat starch, corn starch, etc.); microcrystalline cellulose; celluloses (hydroxypropylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose (HPMC), ethylcellulose, carboxymethylcellulose sodium, etc.); natural gums (gum arabic, alginic acid, guar gum, etc.); liquid glucose, dextrin, povidone, syrup, polyethylene oxide, polyvinylpyrrolidone, poly-N-vinylamide, polyethylene glycol, gelatin, polypropylene glycol, tragacanth gum, etc. be. The amount of binder comprises from about 0.2% to about 14% by weight of the total composition.

Non-limiting examples of glidants include silicon dioxide, colloidal anhydrous silica, magnesium trisilicate, tribasic calcium phosphate, calcium silicate, magnesium silicate, colloidal silicon dioxide, powdered cellulose, starch, such as talc. The amount of glidant is from about 0.01% to about 0.3% by weight of the total composition.

Non-limiting examples of lubricants include magnesium stearate, aluminum stearate, calcium stearate, zinc stearate, stearic acid, polyethylene glycol, glyceryl behenate, mineral oil, sodium stearyl fumarate, talc, hydrogen and modified vegetable oil. The amount of lubricant is from about 0.2% to about 1.0% by weight of the total composition.

The composition can contain a binder that is a low viscosity polymer. Non-limiting examples of low-viscosity polymers include low-viscosity hydroxypropyl methylcellulose polymers such as those sold under the trade name "MethoceLTM" by Dow Chemical (e.g., Methocel E50LVTM, Methocel K100LVRTM, and Methocel F50LVRTM). and a low viscosity hydroxyethyl cellulose polymer. Low viscosity polymers are typically present at a level of from about 10% to about 20%, or from about 10% to about 15%, or preferably about 12%, of the total weight of the composition. Alternatively, in embodiments having a controlled release portion and an immediate release portion, the low viscosity polymer in the controlled release portion is typically at a level of about 15% to about 20%, preferably about 18% by weight of the controlled release portion. exists in

The composition can further include a coating material. Coating materials typically exist as an outer layer on the surface of the dosage form, completely covering the formulation. For example, in some embodiments, the dosage form is an oral tablet in which the controlled release portion forms the first layer of the tablet and the immediate release portion is the second layer deposited over the first layer. to form a core tablet. In such embodiments, for example, the coating material can be in the form of an outer coating layer deposited on the surface of the core tablet. Coating materials typically comprise from about 1% to about 5% by weight of the composition and include hydroxypropylmethylcellulose and/or polyethylene glycol, as well as coating agents, opacifiers, flavoring agents, fillers, abrasives, One or more excipients selected from the group consisting of colorants, anti-caking agents, etc. may be included. Examples of film coating materials and the use of such coating materials are well known to those skilled in the art.

In some embodiments, a salt of a thiazolide compound, or a pharmaceutical composition comprising such a salt, is administered to a mammal (such as a human) within 2 hours or less, or 1.5 hours or less, or 1 hour or less after administration. or 50 minutes or less, or 40 minutes or less, or 30 minutes or less, or 25 minutes or less, or 20 minutes or less, or 15 minutes or less, or 10 minutes or less, or 5 minutes or less. Maximum concentration of compound can be provided. For example, a salt of tizoxanide (such as an amine-containing salt of tizoxanide, which can be, for example, an ethanolamine salt of tizoxanide or a morpholine salt of tizoxanide), or a pharmaceutical composition comprising such a salt, is administered to a mammal (such as a human). 2 hours or less, or 1.5 hours or less, or 1 hour or less, or 50 minutes or less, or 40 minutes or less, or 30 minutes or less, or 25 minutes or less, or 20 minutes or less, or 15 minutes after administration or less, or 10 minutes or less, or 5 minutes or less can provide the maximum concentration of tizoxanide in the plasma of the mammal. In some embodiments, a salt of tizoxanide (such as an amine-containing salt of tizoxanide, which can be, for example, an ethanolamine salt of tizoxanide or a morpholine salt of tizoxanide), or a pharmaceutical composition comprising such a salt, is administered to a mammal. when administered orally to (such as humans), 2 hours or less, or 1.5 hours or less, or 1 hour or less, or 50 minutes or less, or 40 minutes or less, or 30 minutes or less, or 25 minutes or less, or Maximum concentrations of tizoxanide in the plasma of the mammal can be provided in 20 minutes or less, or 15 minutes or less, or 10 minutes or less, or 5 minutes or less.

In some embodiments, a salt of tizoxanide (such as an amine-containing salt of tizoxanide, which can be, for example, an ethanolamine salt of tizoxanide or a morpholine salt of tizoxanide), when administered to a mammal (such as a human), exhibits a It can provide maximum concentrations of tizoxanide in the plasma of mammals faster than nitazoxanide or faster than the same pharmaceutical composition containing nitazoxanide instead of a salt of tizoxanide. In some embodiments, a salt of tizoxanide (such as an amine-containing salt of tizoxanide, which can be, for example, an ethanolamine salt of tizoxanide or a morpholine salt of tizoxanide), when orally administered to a mammal (such as a human): It can provide maximum concentration of tizoxanide in the plasma of the mammal faster than nitazoxanide or faster than the same pharmaceutical composition except containing nitazoxanide instead of a salt of tizoxanide.

In some embodiments, a salt of tizoxanide (such as an amine-containing salt of tizoxanide, which can be, for example, an ethanolamine salt of tizoxanide), when administered to a mammal (such as a human), contains , the AUC _0-12h concentration of tizoxanide and glucoronotizoxanide at or above the AUC _0-12h concentration of nitazoxanide, or at least the AUC _0-12h concentration of the same pharmaceutical composition except containing nitazoxanide instead of a salt of tizoxanide can be provided in In some embodiments, a salt of tizoxanide (such as an amine-containing salt of tizoxanide, which can be, for example, an ethanolamine salt of tizoxanide) is administered orally to a mammal (such as a human) to produce In addition, the AUC _0-12h concentration of tizoxanide and glucoronotizoxanide is equal to or greater than the AUC _0-12h concentration of nitazoxanide, or the AUC _0-12h concentration of the same pharmaceutical composition but containing nitazoxanide instead of a salt of tizoxanide. The above can be provided.

Amine-containing salts of thiazolide compounds have the formula:
can be prepared by reacting a thiazolide compound of (where R is NO ₂ or Cl) with an amine-containing compound (which can be a liquid amine-containing compound) to produce an amine-containing salt of the thiazolide compound.

For such reactions, a thiazolide compound (such as tizoxanide) can be dispersed in a solvent (eg, which can be an alcohol such as methanol or ethanol). An amine-containing compound, which can be a liquid amine-containing compound such as ethanolamine, propanolamine, morpholine, or N-methylpiperazine, can be added to the dispersion. In some embodiments, the temperature of this mixture can be maintained below 30°C, or below 25°C. In some embodiments, the mixture can be agitated. Reaction times can vary. In some embodiments, the reaction time can be 30 minutes to 4 hours, or 1 hour to 3 hours (such as about 2 hours). The mixture can be filtered and the product containing the salt of the amine-containing thiazolide compound washed with a solvent, which can include alcohols such as methanol or ethanol and/or acetate esters such as ethyl acetate. . A product containing a salt of an amine-containing thiazolide compound can be processed under vacuum (pressures less than about 100 mbar, such as 0.2-50 mbar, or 0.5-20 mbar, or 1-10 mbar are possible) and elevated temperature (50° C. ~80C, or 50C to 70C, or 55C to 65C, or any subranges or values within these ranges are possible. In some embodiments, the product containing the salt of the amine-containing thiazolide compound can be dried under vacuum at a temperature of about 15C to about 30C (such as 20C). This dry solid product of the salt of the amine-containing thiazolide compound can be ground and/or crushed.

In some embodiments, when the batch of amine-containing salt produced contains an excess of the amine-containing compound compared to the thiazolide compound, the batch can be purified to eliminate the excess of the amine-containing compound. Such purification can be performed by reslurrying the product in a solvent that can contain an alcohol (such as methanol or ethanol) and/or water. The excess of amine-containing compound compared to thiazolide compound can be determined by measuring the molar ratio of amine-containing compound to thiazolide compound in the batch by a quantitative technique (such as HPLC or LC-MS).

For example, if a batch of the ethanolamine salt of tizoxanide produced contains an excess of ethanolamine relative to the thiazolide, the batch can be purified to eliminate the ethanolamine excess. The excess of ethanolamine to tizoxanide in the batch can be determined by measuring the molar ratio of ethanolamine to tizoxanide in the batch produced by quantitative techniques (such as HPLC or LC-M). For example, a batch may have an excess of ethanolamine to tizoxanide when the molar ratio between ethanolamine and tizoxanide is greater than 1.00, or greater than 1.05. If judged to be in excess, after purification, the batch produced has a molar ratio between ethanolamine and tizoxanide of 0.9 to 1.00, or 0.95 to 1.00, or 0.96 to 1.00, or 0.97 to 1.00, or 0.98 to 1.00, or 0.99 to 1.00.

Thiazolide compounds (such as tizoxanide) can be generated from their respective prodrugs. For example, tizoxanide can be obtained by heating a solution containing nitazoxanide to a first elevated temperature (at least 50°C, or at least 55°C, or at least 60°C, or at least 65°C, or at least 70°C, or at least 75°C). can be prepared. The solvent in the solution can be a polar solvent such as dimethylacetamide or dimethylformamide. In some embodiments, nitazoxanide can be dispersed in a solvent to form a solution. In some embodiments, an acid (such as HCl, which can be about 0.5M to 3M (such as 1M) dilute acid (such as HCl)) can be added to the solution. After addition of the acid, in some embodiments, the temperature of the mixture can be further increased to a second elevated temperature, such as at least 65°C, or at least 70°C, or at least 75°C. In some embodiments, each of the first and second elevated temperatures can be 100° C. or less, or 95° C. or less, or 90° C. or less, or 85° C. or less, or 80° C. or less. Heating at the second elevated temperature can continue for at least 10 hours, or at least 15 hours, or at least 20 hours, or at least 25 hours, or at least 30 hours. In some embodiments, heating at the second elevated temperature is for 10 hours to 70 hours, or 15 hours to 65 hours, or 20 hours to 60 hours, or 30 hours to 50 hours, or within these ranges. can continue over any value or subrange of . After conversion of nitazoxanide to tizoxanide, the solution can be cooled, for example, to room temperature (such as about 25° C.) and neutralized with a base (such as KOH or NaOH, such as about 1 M NaOH). The temperature can be maintained below 30°C, or below 25°C during neutralization. Tizoxanide formed from nitazoxanide can be used for subsequent salt formation without drying.

In some embodiments, the conversion of nitazoxanide to tizoxanide is accomplished without the use of concentrated acids and/or concentrated bases; without handling strongly acidic or alkaline mixtures based on any parameter); and/or volatile It can be carried out without the use of solvents (such as solvents with boiling points below 100°C).

In some embodiments, tizoxanide can be prepared from nitazoxanide by preparing an aqueous solution of ammonia in tetrahydrofuran, then evaporating, suspending in cold acid (such as cold aqueous HCl), and filtering. Prepared by

Methods for converting nitazoxanide to tizoxanide are also disclosed, for example, in Rossignol and Stachulski, J. Chem. Res. (S), 1999, 44-45, which is hereby incorporated by reference in its entirety.

Salts of thiazolide compounds (such as tizoxanide or RM-4848), and pharmaceutical compositions comprising such salts, are the same ones for which nitazoxanide, tizoxanide, and/or RM-4848 are known to be useful. It may be useful for the above purposes. For example, the salts or pharmaceutical compositions thereof can be used for administration to a subject (such as a human) to treat a disease or disorder potentially treatable with nitazoxanide or tizoxanide, which disease or disorder is, for example, influenza infection, Influenza-like illnesses, respiratory infections, diseases or conditions caused by viruses belonging to the genus Enterovirus (such as rhinoviruses and/or enteroviruses), diseases or conditions caused by viruses belonging to the coronavirus family (such as coronaviruses), paramyxoviruses diseases or conditions caused by viruses belonging to the family (such as respiratory syncytial virus, Sendai virus, or Hendra virus), hepatitis C, hepatitis B (including chronic hepatitis B), intestinal parasitic infections, These include diarrhea caused by Ptosporidium parvum and Giardia lamblia. For therapeutic purposes, a salt of a tizolid compound (such as tizoxanide) is administered to a subject (such as a human) in a therapeutically effective amount (a disease amount, which is a disease or disorder potentially treatable with nitazoxanide and/or tizoxanide). is sufficient to ameliorate one or more symptoms).

In some embodiments, the amine-containing salt of tizoxanide can be the ethanolamine salt of tizoxanide. In some embodiments, such salts are in the form of particles having an average size of 50 microns or less, or 45 microns or less, or 40 microns or less, or 30 microns or less, or 25 microns or less, or 20 microns or less. be able to. In some embodiments, the ethanolamine salt of tizoxanide can contain microparticles, wherein at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the particles have about 1 microns to about 60 microns, or about 2 microns to about 50 microns, or about 3 microns to about 45 microns, or about 4 microns to about 40 microns, or about 4 microns to about 35 microns, or about 4 microns to about 30 microns , or with any value or subrange size within these ranges. In some embodiments, the ethanolamine salt of tizoxanide optionally further has a size of at least about 100 microns (such as from about 100 microns to about 2000 microns, or from about 100 to about 1500 microns, or from about 100 to about 1000 microns). 30% or less, or 20% or less, or 10% or less of coarse particles having In some embodiments, a batch of ethanolamine salt of tizoxanide is prepared from a batch of tizoxanide such that the batch of ethanolamine salt of tizoxanide has an average particle size and/or particle distribution that distinguishes the batch of tizoxanide. be able to.

In some embodiments, the ethanolamine salt of tizoxanide can have a melting point of about 144C to about 150C or about 146C to about 148C.

In some embodiments, the ethanolamine salt of tizoxanide can be in crystalline form.

In some embodiments, the ethanolamine salt of tizoxanide can have a differential scanning calorimetry (DSC) curve like FIG. 12A.

In some embodiments, the ethanolamine salt of tizoxanide can have a thermogravimetric analysis (TGA) thermogram such as FIG. 11A.

In some embodiments, the ethanolamine salt of tizoxanide can have an X-ray powder diffractogram determined with a diffractometer using Cu—Kβ radiation at a wavelength of 1.39222 Å, as in FIG. 25A.

In some embodiments, the ethanolamine salt of tizoxanide can have an X-ray powder diffractogram determined with a diffractometer using Cu—Kβ radiation with a wavelength of 1.39222 Å, wherein the diffractogram is 1 It has one or more peaks at about 8.5°, about 11.2°, about 16.8°, about 19.5°, about 20.9°, about 25.6°, about 27.0°, and about 36.1° two-theta.

In some embodiments, the ethanolamine salt of tizoxanide can have an X-ray powder diffractogram determined with a diffractometer using Cu—Kβ radiation with a wavelength of 1.39222 Å, wherein the diffractogram is 1 8.5°±0.2°, 11.2°±0.2°, 16.8°±0.2°, 19.5°±0.2°, 20.9°±0.2°, 25.6°±0.2°, 27.0°±0.2°, and 36.1° Hold at a position of ±0.2° 2θ.

In some embodiments, the ethanolamine salt of tizoxanide can have an X-ray powder diffractogram determined with a diffractometer using Cu-Kβ radiation with a wavelength of 1.39222 Å, the diffractogram comprising a peak 8.5°±0.2°, 11.2°±0.2°, 16.8°±0.2°, 19.5°±0.2°, 20.9°±0.2°, 25.6°±0.2°, 27.0°±0.2°, and 36.1°±0.2° 2θ position.

In some embodiments, the ethanolamine salt of tizoxanide can be in batch form. Such batches contain at least 0.1 kg, or at least 0.2 kg, or at least 0.3 kg, or at least 0.4 kg, or at least 0.5 kg, or at least 0.6 kg, or at least 0.7 kg, or at least 0.8 kg, or at least 0.9 kg, or at least 1.0 kg, or at least 1.2 kg, or at least 1.5 kg, or at least 2.0 kg, or at least 2.3 kg, or at least 2.5 kg, or at least 3.0 kg, or at least 4 kg, or at least 5 kg, or at least 7 kg, or at least 10 kg, or at least 15 kg, or at least 20 kg, or at least 25 kg, or at least 30 kg, or at least 35 kg, or at least 40 kg of the ethanolamine salt of tizoxanide. In some embodiments, the molar ratio between ethanolamine and tizoxanide within such batch is between 0.9 and 1.00, or between 0.95 and 1.00, or between 0.96 and 1.00, or between 0.97 and 1.00. , or between 0.98 and 1.00, or between 0.99 and 1.00. The molar ratio of ethanolamine to tizoxanide in batches of ethanolamine salts can be determined by a number of techniques, including high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). be.

In some embodiments, the ethanolamine salt of tizoxanide can be prepared by reacting tizoxanide with ethanolamine. Tizoxanide to be reacted with ethanolamine can be prepared in advance from nitazoxanide. Thus, in some embodiments, the ethanolamine salt of tizoxanide is prepared by a process comprising two steps: Step 1: preparation of tizoxanide from nitazoxanide; It can be prepared by a method comprising preparation.

The embodiments described herein are further illustrated by, but not limited to, the following examples.

Example 1. 2-Hydroxybenzoyl-N-[(5-nitro)thiazol-2-yl]amide, ethanol salt. (RM-5071)

Tizoxanide (i.e., 2-hydroxybenzoyl-N-[(5-nitro)thiazol-2-yl]amide, 0.53 g, 2 mmol) was suspended in methanol (MeOH, 20 ml) containing ethanolamine (0.15 mL). made it cloudy. The suspension was warmed to 50° C. over a few minutes to give a virtually clear yellow solution that readily began to crystallize when filtered and concentrated to 5 mL. Diethyl ether (Et ₂ O, 5 mL) was added and the mixture cooled to 0° C. for complete crystallization. Filtration and washing with Et ₂ O containing a small amount of MeOH gave the title salt 1 (0.49 g, 75%) as a yellow crystalline solid; mp: 158-160° C. (decomposition); H, 4.2; N, 17.35; S, 9.8. _{C12H14N4O5S requires} C, _44.2 ; H _, 4.3; N, _17.2 ; S, 9.8% _{; CH2CH2 )} , 3.57 (2H, t, _CH2CH2 ) _, 5.20 (1H, brs, OH), 6.81 (2H, m, ArH), 7.32 (1H, m, ArH), 7.67 (3H, br s, NH ₃ ⁺ ), 7.91 (1H, m, ArH), 8.51 (1H, s, 4′-H), and 14.71 (1H, br s, NH); C NMR [100 MHz, (CD ₃ ) ₂ SO] δ 41.6, 57.9, 117.5, 118.2, 119.9, 130.1, 133.4, 137.9, 145.9, 161.3, 171.6, and 172.2; m/z (-ve ion electrospray mode) 264 [(MH) ^? ]. Measured value: m/z, 264.0092. _C10H6N3O4S claims _{m /} z, _264.0085 .

Example 2. 2-Hydroxybenzoyl-N-[(5-chloro)thiazol-2-yl]amide, ethanol salt 2

This salt was prepared analogously to the salt of Example 1 using RM4848, 2-hydroxybenzoyl-N-[(5-chloro)thiazol-2-yl]amide (0.51 g, 2 mmol), resulting in Product 2 was obtained (0.48 g, 76%); found: C, 45.7; H, 4.5; N, 13.35; S, 10.15. _{C12H14ClN3O3S} requires C, 45.6; _H , 4.5; _N , _13.3 ; _S , 10.15% _; , _{CH2CH2 )} , 3.58 (2H, t, _CH2CH2 ), 5.20 (1H, brs, OH), 6.67-6.73 (2H _, 2m, ArH), 7.20 (1H, m, ArH ), 7.23 (1 H, s, 4′-H), and 7.83 (1 H, dd, ArH); NH ₃ ⁺ appears as a very broad signal centered at δ 7.65; 13C NMR [100 MHz, ( _CD3 ) _2SO ] δ 41.7, 58.0, 114.3, 116.8, 117.4, 120.4, 129.4, 132.1, 135.3, 162.4, 164.5, and 169.5; m/z (-ve ion electrospray mode) 253 [(MH) ^? ]. Measured value: m/z, 252.9849. _{C10H635ClN2O2S claims m} /z _, ^252.9844 .

Example 3. 2-Hydroxybenzoyl-N-[(5-nitro)thiazol-2-yl]amide, morpholine salt 3 (RM-5072)

This salt was prepared analogously to the salt of Example 1 by adding tizoxanide (2-hydroxybenzoyl-N-[(5-nitro)thiazol-2-yl]amide; 0.53 g, 2 mmol) and morpholine (0.24 mL). From 3 was obtained as a yellow microcrystalline solid (0.66 g, 94%); found: C, 47.7; H, 4.6; N, 16.0; S, 9.2. _{C14H16N4O5S requires} C, _47.7 ; H _, 4.6; N, _15.9 ; _S , 9.1%; , 2m, 2xCH ₂ CH ₂ ), 6.82 (2 H, m, ArH), 7.31 (1 H, t, ArH), 7.91 (1 H, m, ArH), and 8.51 (1 H, s, 4´- H); 13C NMR [100 MHz, ( _CD3 ) _2SO ] ?

Example 4. 2-Hydroxybenzoyl-N-[(5-chloro)thiazol-2-yl]amide, morpholine salt 4

This salt was prepared analogously to the salt of Example 1 from RM4848 (2-hydroxybenzoyl-N-[(5-chloro)thiazol-2-yl]amide; 0.51 g, 2 mmol) and morpholine (0.24 mL). did. In this case the first solid that separated was unchanged RM4848; concentration of the mother liquor gave the desired salt 4 (0.198 g, 29%); found: C, 49.4; H, 4.9; , 12.2; S, 9.2. _{C14H16ClN3O3S requires} C, _49.2 ; H, 4.7; N, _12.3 ; _S , 9.4 _% ; ), 3.71 (4 H, m), 6.70 (2 H, 2m, ArH), 7.21 (1 H, t, ArH), 7.24 (1 H, s, 4´-H), and 7.83 (1 H, d , ArH); 13C NMR [100 MHz, ( _CD3 ) _2SO ] ?

Example 5. 2-Hydroxybenzoyl-N-[(5-nitro)thiazol-2-yl]amide, propanol salt

This was prepared analogously to the salt of Example 1 from tizoxanide (0.53 g, 2 mmol) and 3-amino-1-propanol (0.19 mL) to give the desired salt 5 as orange crystals (0.395 g, 58%). Found: C, 5.9; H, 4.7; N, 16.4; S, 9.5. _C13H16N4O5S requires _C , 45.9; H _, 4.7; N, _16.5 ; _{S ,} 9.4% _; m, _{CH2CH2CH2 )} , 2.86 (2H, t, _CH2CH2 ), 3.30 (1H, brs, _OH ), 3.49 (2H _, t, _CH2CH2 ), _6.81 (2 H, m, ArH), 7.30 (1 H, t, ArH), 7.55 (3 H, br s, _NH3 ⁺ ), 7.90, (1 H, d, ArH), 8.51 (1 H, s, 4´ -H), and 14.71 (1 H, s); C NMR [100 _MHz , ( _CD3 ) _2SO ] ? 171.6, and 172.2.

Example 6. 2-Hydroxybenzoyl-N-[(5-chloro)thiazol-2-yl]amide, 3-amino-1-propanol salt

This was done analogously to 1 from RM4848 (2-hydroxybenzoyl-N-[(5-nitro)thiazol-2-yl]amide; 0.51 g, 2 mmol) and 3-amino-1-propanol (0.16 mL). prepared. The final methanol solution was concentrated and diluted with Et ₂ O to crystallize; after cooling the mixture to complete crystallization, the solid was filtered, washed with Et ₂ O and dried to give the desired salt. 6 was obtained (0.51 g, 77%). Found: C, 47.5; H, 5.0; N, 12.7; S, 9.6. _{C13H16ClN3O3S requires} C, 47.3; H, 4.9; _N , _{12.7 ;} S, _9.7 % _; m, _{CH2CH2CH2 )} , 2.86 (2H, t, _CH2CH2 ), 3.49 (2H _, t, _CH2CH2 ), _6.70 (2H, m, ArH ₎ , 7.19 (1H , dt, ArH), 7.22 (1 H, s, 4'-H), and 7.83 (1 H, dd, ArH); C NMR [100 MHz, ( _CD3 ) _2SO ] ? _C 30.5, 37.3, 58.4 , 114.2, 116.8, 117.3, 120.4, 129.4, 132.0, 135.3, 162.3, 164.5, and 169.5.

Example 7. 2-Hydroxybenzoyl-N-[(5-nitro)thiazol-2-yl]amide, diethanolamine salt

Tizoxanide (i.e. 2-hydroxybenzoyl N-[(5-nitro)thiazol-2-yl]amide, 0.53 g, 2 mmol) was suspended in methanol (MeOH, 70 ml) containing diethanolamine (0.20 mL). was warmed until an almost complete solution was obtained and then filtered. After cooling the clear filtrate was concentrated and then cooled to 0° C. to allow complete crystallization. After filtration, washing with Et ₂ O containing a small amount of MeOH and drying gave the desired product in two crops to give the title salt 7 as a yellow crystalline solid (0.36 g, 49%). . Found: C, 45.35; H, 4.9; N, 15.25; S, 8.7. _{C14H18N4O6S requires} C, 45.4; H, 4.9; _N , _15.25 ; _S , 8.7 _{% ;} , _{2xCH2CH2 )} , 3.66 (4H, t, _2xCH2CH2 ), 5.18 (2H, brs, OH), 6.81 (2H _, m, ArH), 7.31 (1H, m, ArH), 7.91 (1H , m, ArH), 8.34 (2H, br s, NHs), 8.50 (1 H, s, 4´-H), and 14.70 (1H, br s, NH); C NMR [100 MHz, ( _CD3 ) ₂ SO] δ _C 49.3, 56.7, 117.5, 118.2, 119.9, 130.1, 133.4, 137.9, 145.9, 161.3, 171.6, and 172.

Example 8. 2-Hydroxybenzoyl-N-[(5-chloro)thiazol-2-yl]amide, diethanolamine salt

RM4848 (2-hydroxybenzoyl-N-[(5-chloro)thiazol-2-yl]amide; 0.51 g, 2 mmol) and diethanolamine (0.24 mL) in hot MeOH (30 mL) Prepared similarly. After removing a small amount of insoluble material by filtration, the filtrate was concentrated and _Et2O was added. After cooling the mixture to complete crystallization, the solid was filtered off, washed with Et ₂ O containing a small amount of MeOH and dried to give the title salt 8 as an almost white solid. (0.545 g, 76%). Found: C, 46.9; H, 5.1; N, 11.8; S, 8.85. _{C14H18ClN3O4S requires} C _, 46.7; H, 5.0 _; N, _11.7 ; _S , 8.9 _% ; t, _{2xCH2CH2 )} , 3.65 (4H, t, _2xCH2CH2 ), 6.70 (2H, m, _ArH ), 7.20 (1H, m, ArH), 7.23 (1H, s, 4´ -H), and 7.84 (1 H, dd, ArH); broad interchangeable peaks at δ 5.17 (2 H) and δ 8.2; 13C NMR [100 MHz, (CD ₃ ) ₂ SO] δ _C 49.4, 56.8, 114.4, 116.9, 117.4, 120.3, 129.5, 132.2, 135.3, 162.2, 164.2, and 169.3.

Example 9. 2-Hydroxybenzoyl-N-[(5-nitro)thiazol-2-yl]amide, N-methylpiperazine salt

Tizoxanide (0.50 g, 1.90 mmol, 1.0 eq) was suspended in methanol (10 mL) along with N-methylpiperazine (0.90 mL). Additional methanol (10 mL) was added to the mixture and heated to produce a clear solution. The mixture was then left overnight. The resulting precipitate was filtered off to give the final product as a yellow crystalline solid (0.13 g, 19% yield). Found: C, 49.0; H, 5.2; N, 19.0; S, 9.0. _C15H19N5O4S requires C _, 49.3; _{H ,} 5.2; N, _19.2 ; _S , 8.8% _; ), 2.50-2.51 (4H, m), 3.06-3.09 (4H, m), 6.80-6.84 (2H, m), 7.32 (1H, t, J = 7.7 Hz), 7.92 (1H, d, J = 8.1 Hz), 8.52 (1h, s); 13c NMR [400 MHz, (CD ₃ ) ₂ So] Δ _C 43.36, 45.79, 517.48, 118.20, 119.85, 119.85, 130.07, 133.38, 137.95, 145.91, 171.50. And 172.16.

Example 10. A Bioavailability Pharmacokinetic Study of RM-5071 and RM-5072 Orally Administered to Rats

summary

Tizoxanide (T ) and tizoxanide glucuronide (TG) bioavailability. Plasma samples were collected at 0.083, 0.167, 0.25, 0.5 1, 2, 6, 12, and 24 hours after dosing. Mass spectrometry was used to determine the concentrations of T and TG. No adverse clinical signs were observed in any of the three groups of rats. Both RM-5071 and RM-5072 dramatically improve plasma T and TG availability rates compared to NTZ. These compounds are rapidly absorbed after oral administration, reaching C _max within 5 minutes. RM-5071 was associated with greater plasma concentrations of T and TG and less variability in absorption compared to RM-5072 or NTZ.

Introduction

Nitazoxanide (NTZ), a prodrug for T and TG, is poorly absorbed after oral administration in animals and humans. Absorption is markedly influenced by diet, and there is significant intrasubject and intersubject variability in T and TG concentrations. Two new salts of T, namely RM-5071 and RM-5072, were prepared and evaluated for their potential to improve the bioavailability of T and TG after oral administration. This study was conducted to demonstrate the biological effects of T and TG in plasma following oral administration of a single dose of 90 mg/kg RM-5071, RM-5072, and NTZ by oral gavage to Sprague-Dawley rats. Evaluate availability.

Materials and methods

RM-5071 is 2-hydroxybenzoyl-N-[(5-nitro)thiazol-2-yl]amide, ethanolamine salt. RM-5072 is 2-hydroxybenzoyl-N-[(5-nitro)thiazol-2-yl]amide, morpholine salt. RM-5071 and RM-5072 were as disclosed above.

animals and treatment. Three groups of 4 male and 4 female Sprague-Dawley rats were given a single gavage dose of RM-5071, RM-5072, or NTZ as detailed in the table below. was administered as

Nine serial blood samples were obtained from each animal at 0.083, 0.167, 0.25, 0.5 1, 2, 6, 12, and 24 hours after dosing. Derived plasma samples were stored at -70°C or below in sodium heparinized tubes until sent on dry ice by overnight delivery for analysis of T and TG concentrations by mass spectrometry.

result

No adverse clinical signs were observed in any of the three groups of rats.

After administration of RM-5071, RM-5072, and NTZ, the maximum concentration (Cmax) of T (median) was 4.7, 3.1, and 1.7 μg/mL, respectively. RM-5071 and RM-5072 reached Cmax at 5 minutes post-dose, at the time of the first plasma sampling. In the case of NTZ, the plasma T _Cmax (only 1.7 μg/mL) was reached after 2 hours.

Since some animals become glucuronidated T faster than others, the sum of free T and glucuronidated T concentrations at each time point was used to determine the extent, rate, and variability of absorption of these three compounds. evaluated the sex. To arrive at the concentration of glucuronidated T, the concentration of TG was multiplied by 61% (MW of T divided by MW of TG = 441 = 270).

The sum of the median concentrations of free T plus glucuronidated T in plasma over 12 hours after dosing is shown in FIG.

Mean _Cmax and AUC _0-12h values for the sum of free T and glucuronidated T are shown in Table 2 along with the relative standard deviation (RSD). The mean _Cmax of RM-5071 was 33% and 47% higher than RM-5072 and NTZ, respectively, and the RSD was 31% compared to 44% for both RM-5072 and NTZ.

1 relative standard deviation

The mean AUC _0-12h for RM-5071 was almost double that of RM-5072, but about the same as that of NTZ. Comparison of AUC _0-12h with NTZ is due to the collection of only one plasma sample (6 hour sample) between 2 and 12 hours and the fact that NTZ is absorbed more slowly than other compounds. to be influenced. It is likely that the actual AUC _0-12h values for NTZ would have been much smaller if additional samples had been collected, especially at time points between 6 and 12 hours after dosing.

Of note, the RSD associated with the mean AUC _0-12h for RM-5071 was only 16% compared to 36% for RM-5072 and NTZ. This indicates that the inter-subject variability of absorption associated with NTZ is significantly ameliorated by RM-5071.

Conclusion:

Both RM-5071 and RM-5072 dramatically improve the rate of T availability in plasma compared to NTZ. These compounds are rapidly absorbed, reaching Cmax within 5 minutes of oral administration. RM-5071 is associated with greater plasma concentrations of free T and glucurono-T and less variability in absorption compared to either RM-5072 or NTZ. This study shows that the rate, extent, and mutability of absorption are improved with RM-5071 compared to RM-5072 or NTZ.

Example 11. amine salt of thiazolide

A total of 10 amine salts (5 each for tizoxanide and RM4848) were made.
1: Ethanolamine:
2: 3-aminopropan-1-ol (propanolamine):
3: Morpholine:
4: Piperazine:
5: N-methyl piperazine:
Tizoxanide:
RM-4848:

General procedure

The appropriate amine is heated in methanol with an equal molar amount of tizoxanide or RM4848 until a clear solution is obtained. Any small undissolved solids are removed by filtration. Upon cooling, the desired salt may crystallize immediately; (for the tizoxanide salt) the addition of an equal volume of solvent (such as diethyl ether) or (for the RM4848 salt) the addition of excess water after concentration to a small volume. Addition of ether can be used to obtain a solid product. In general, RM4848 salts are more soluble under the above conditions. All salts are more water soluble than the parent thiazolide.

1: Both tizoxanide and RM-4848 readily formed crystalline salts with ethanolamine (RM5071 and RM5072). They were obtained in good crystalline form and microanalytical pure. The phrase "microanalytical pure" can mean that the amount of each atom in the synthesized molecule deviates from each theoretical value within ±0.3% of the theoretical value.

2: Essentially the same as for ethanolamine. Excellent yield, crystalline morphology, and chemical purity for both propanolamine salts.

3: The morpholine salt of tizoxanide was readily obtained in high purity. In the case of RM4848, the first compound isolated as a solid was unchanged RM4848. Concentration of the mother liquor to a small volume gave the desired salt in about 30% yield but with high purity.

4: Both tizoxanide and RM4848 piperazine salts were obtained by standard methods, but they were difficult to obtain in satisfactory purity. Excess piperazine appears to crystallize with salt. Although the invention is not limited by its principle of operation, this may be due to the fact that piperazine itself is a solid.

5: Using liquid N-methylpiperazine gave tizoxanide salt in very good yield and purity. The corresponding salt of RM4848 was obtained as a solid, but less pure.

Synthesis of RM-5071 from nitazoxanide

Step 1: Preparation of tizoxanide from nitazoxanide

Nitazoxanide was dissolved in a polar solvent such as 3Veq of DMF. The solution was heated to an elevated temperature (such as 50°C). Acid (such as HCl 1M) was added at eg 1 Veq. The solution was further heated to a second elevated temperature (such as 70°C) to effect complete conversion. It can take a period of about 36 hours to about 48 hours. The solution was cooled to room temperature and neutralized with a base (such as 1M NaOH). The solution was filtered and the resulting cake was washed with a solvent such as water and/or an alcohol such as methanol. This reaction allows 90-100% recovery of tizoxanide with good purity. This reaction is suitable for upscaling.

Step 2: Preparation of RM-5071 from Tizoxanide

Tizoxanide was dispersed in, for example, 5 Veq of a solvent (can be an alcohol such as methanol) at room temperature. Ethanolamine was added slowly, eg 1.1 equivalents. Fever. The mixture is stirred for about 2 hours, for example. The mixture was then filtered and the resulting cake was washed with a solvent such as methanol and ethyl acetate, which can be about a 1:1 volume ratio. The cake was dried to a solid under vacuum (such as less than about 100 mbar) at elevated temperature (such as about 60°C). The dried solid can be ground and/or crushed. This reaction allows 80-90% recovery of RM-5071 with good purity. This reaction is suitable for upscaling.

summary

RM-5071 can be synthesized from nitazoxanide in a two-step synthesis. The conditions for manufacturing RM-5071 can be adapted for upscaling in manufacturing facilities. This is because manufacturing facilities utilize limiting dilution, mild conditions, and product recovery by centrifugation. Products are usually obtained in good purity. Two purification possibilities in case of poor purity. Yields are possible of 80-85 g of RM5071 from 100 g of nitazoxanide. A typical upscaling condition could be:

Analytical information about RM-5071

Example 12

summary

Nitazoxanide (NTZ), a thiazolide typified by 2-[(acetyloxy)-N-(5-nitro-2-thiazolyl)]benzamide, is an important class of multipharmacological agents with a broad spectrum of anti-infective activity. may have A prototype NTZ, originally marketed as an antiparasitic agent specifically against Cryptosporidium species, was subsequently shown to be effective against a number of viruses. However, the pharmacokinetic parameters of NTZ are not ideal due to its low solubility and absorption when efficient systemic circulation is required. This study reports the preparation and evaluation of a series of amine salts of tizoxanide, the active deacetyl metabolite of NTZ, and the corresponding 5-Cl thiazolide RM4848. Thiazolide salts have recently been scaled up for clinical trials as they have demonstrated improved water solubility and absorption as shown by in vivo measurements.

Introduction

Nitazoxanide [NTZ; 2-[(acetyloxy)-N-(5-nitro-2-thiazolyl)]benzamide] ^1a was first reported by Rossignol and Cavier in 19761; it is a known anti-infective agent. It is modeled on the basis of a niclosamide 2 ² , with the anilide replaced by a nitrothiazolylamide, and has shown promising antiparasitic activity in vitro and in vivo. NTZ 1a was originally developed for the treatment of parasitic infections ^by protozoa and helminths3-5, but later its most important use was in the treatment ^of Cryptosporidium spp ^. Until now, it is the only FDA-approved treatment for Cryptosporidium parvum. Studies of its antiparasitic activity have established that one important mode of action of NTZ 1a is inhibition of the folding chaperone protein disulfide isomerase. ⁸ NTZ 1a also has valuable antibacterial activity against both aerobic and anaerobic species, acting in the case of anaerobic ^{bacteria by inhibiting pyruvate oxidoreductase 9,10 .}

NTZ 1a was found to have antiviral activity while treating cristoporidiosis in AIDS patients ¹¹ . The first clinical trials using NTZ 1a as an antiviral agent were against rotavirus-induced diarrhea ¹² , and included children as patients. NTZ 1a proved to have antiviral activity against a number of viruses ^13-16

The nitro group may not be essential for activity: the 5'-Cl analogue 3a may have a similar spectrum of activity at small micromolar values, ¹⁸ and the 4'-ethanesulfonyl analogue 4 may be active against influenza A. IC ₅₀ =0.14 μM ^19-21 , demonstrating excellent in vitro activity against the H1N1 strain of virus.

Although NTZ 1a is normally administered orally, only a small portion is absorbed from the gastrointestinal tract ²² . It is actually a prodrug of the deacetyl derivative tizoxanide 1b, formed shortly after absorption and then excreted from the body mostly as O-glucuronide ⁵²³ :1a has a plasma half-life of 1.3 hours. Although such a biodisposition may be acceptable in intestinal infections, it may be difficult to achieve adequate systemic circulation of 1a/1b due to viral infections (such as influenza A). have a nature. Prodrug amino acid esters (such as 6) that may improve the absolute oral bioavailability of 1a by about 20% in the case of 6 ²⁴ .

Another approach to improve the pharmacokinetic parameters of 1a/1b is to administer the active agent as an amine salt.

This study reports the synthesis, their characterization, and selected pharmacokinetic data of a series of amine salts of thiazolide 1b and RM4848 3b. In general, not all the amines we selected gave satisfactory results and the behavior of 1b and 3b differed in some cases.

consideration

Heating tizoxanide 1b in methanol with a slight excess of ethanolamine for about 0.25 hours gave an almost clear solution. Filtration followed by concentration crystallized the desired salt 8; after dilution with diethyl ether, cooling and filtration, 8 was obtained in good yield and good microanalytical purity. 1H NMR showed a characteristic upfield shift of the aryl proton. This is consistent with the anionic nature of thiazolides. Similarly, salt 9 was obtained from RM4848 3b. Figure 4 summarizes a total of nine salts similarly made with hydroxylamine, morpholine, and N-Me piperazine.

Some significant differences were seen with certain thiazolide/amine combinations. For example, the morpholine salt 10 was obtained from 1b in the usual manner, but when RM4848 3b was used, the first solid that precipitated was unreacted 3b. Concentration of the filtrate gave the desired salt 11, which was inevitably in rather low yield, but was still microanalytical pure. Salts 12-15 were similarly obtained using 1-aminopropanol (12, 13) and diethanolamine (14, 15). The pure salt could not be readily obtained from the piperazine as the diamine proved to be more difficult to handle; the piperazine is a solid and difficult to remove by recrystallization. However, N-Me piperazine (liquid) and 1b gave the salt 16 in low yield; in this case the site of protonation was not defined. The amino acid L-lysine did not give a separable salt from 1b or 3b.

upscaling

The synthesis of ethanolamine salt 8 was successfully scaled up to an industrial process. It is a two-step synthesis (80% yield) starting from the FDA-approved drug nitazoxanide 1a. A pre-technical batch of 40 kg of pure material was prepared. Production plant equipment has been utilized to demonstrate that the process is reliable for large scale manufacturing.

Pharmacokinetics

Pharmacokinetic data are presented in Example 10.

experiment

General experimental method

Salts were prepared as outlined in Examples 1-9.

1H and 13C spectra were acquired on a Bruker 400 MHz instrument equipped with a multinuclear 5-mm BBFO probe (100 MHz for 13C spectra). Spectra of 1H (at 400.13 MHz) and 13C(1H) (at 100.61 Mhz) were acquired at ambient temperature using standard parameter sets; solvent resonances were used for reference purposes.

Low resolution was obtained by injecting the sample solution directly into a Micromass LCT mass spectrometer (Micromass LCT Waters Micromass UK Ltd, Manchester, UK) operating in electrospray mode with +ve ions or -ve ions as indicated. and obtained high-resolution mass spectra.

References

1. J.-F. Rossignol and R. Cavier, Chem. Abstr., 1975, 83, 28216n.
2. GJ Frayha, et al, Gen. Pharmacol., 1997, 28, 273-299.
3. R. Cavier and J. -F. Rossignol, Rev. Med. Vet., 1982, 133, 779-783.
4. LM Fox and LD Sarvolatz, Clin. Infect. Dis., 2005, 40, 1173-1180.
5. J. -F. Rossignol and H. Maisonneuve, Am. J. Trop. Med. Hyg., 1984, 33, 511-512.
6. O. Doumbo, et al., Am. J. Trop. Med. Hyg., 1997, 56, 637-639.
7. J. -F. Rossignol, A. Ayoub and MS Ayers, J. Infect. Dis., 2001, 184 103-106.
8. J. Muller, et al., Exp. Parasitol., 2008, 118, 80-88.
9. L. Dubreuil, et al., Antimicrob. Agents Chemother., 1996, 40, 2266-2270.
10. PS Hoffman, et al., Antimicrob. Agents Chemother., 2007, 51, 868-876.
11. J. -F. Rossignol, Aliment. Pharmacol. Ther., 2006, 24, 887-894.
12. J.-F. Rossignol, et al., Lancet, 2006, 368, 124-129.
13. J.-F. Rossignol and EB Keefe, Future Microbiol., 2008, 3, 539-545.
14. J. -F. Rossignol, et al, Aliment. Pharmacol. Ther., 2008, 28, 574-580.
15. J. Haffizulla, et al., Lancet Infect. Dis., 2014, 14, 609-618.
16. J. -F. Rossignol, Antiviral Res., 2014, 110, 94-103.
17. NIH Website clinicaltrials.gov NCT04341493.
18. BE Korba, et al., Antiviral Res., 2008, 77, 56-63.
19. AV Stachulski, et al J. Med. Chem., 2011, 54, 4119-4132.
20. AV Stachulski, et al., J. Med. Chem., 2011, 54, 8670-8680.
21. AV Stachulski, et al., Future Med. Chem., 2018, 10, 851-862.
22. J. Broekhuysen, et al. Int. J. Clin. Pharmacol.
23. J. -F. Rossignol and AV Stachulski, J. Chem. Res. (S), 1999, 44-45.
24. AV Stachulski, et al., Eur. J. Med. Chem., 2017, 126, 154-159.
25. G. Hecht and C. Gloxhuber, Z. Tropenmed. Parasit., 1962, 13, 1-8.
26. P. Andrews, et al., Pharmac. Ther., 1983, 19, 245-295.
27. R. Krieger and W. Krieger, Handbook of pesticide toxicology, 2001, 1225-1247.
28. H. Tao, et al, Nat. Med. 2014, 20, 1263-1269.
29. D. Lu, et al., Xenobiotica, 2016, 46, 1-13.
30. MA Gemmell, et al., Res. In Vet. Sci., 1977, 22, 389-391.

Additional references

Rossignol, JF Antiviral Res 2014; 110:94-103.
Wang, M., et al. Cell Research (2020) 0:1-3; https://doi.org/10.1038/s41422-020-0282-0
Rossignol, JF & Van Baalen, C. Poster presentation at the 2nd International Meeting on Respiratory Pathogens March 7-9, 2018, Abstract ARP057 page 19
Braakman, I., et al. Nature 1992; 356:260-62
Braakman, I., et al. J. Cell Biol. 1991; 114:401-11
Doms. RW, et al. J. Cell Biol 1987:105:1957-69
Chang, CW, et al., J. Biomedical Sci 2009; 16:80
Mirazimi A, Svensson L VP7 J Virol, 2000; 74: 8048-52
Rossignol, JF, et al., Journal of Biological Chemistry. 2009; 284:29798-29808.
Piacentini S, et al., Research Report, 2018;8: 10425
Cao J, Forest C, Zhang X Antiviral Res, 2015; 114: 1-10
Lee, JH, et al., Int J Obes 2017; 41: 645-51
Sag, D., et al., J. Immunol 2008; 181:8633-41
Wang, W., et al., J. Biol Chem 2003: 278:27016-23
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RA Mook, et al., Bioorg. Med. Chem., 2015, 23, 5829-5838.
A. Jurgelt, et al., PLOS Pathogens, 2012, 8, e1002976.

Example 13

RM5071 is a prodrug of tizoxanide (TIZ), the active metabolite of nitazoxanide (NTZA), an antiprotozoan drug called Alinia, approved by the FDA. RM5071 is an organic salt consisting of two parts, tizoxanide and ethanolamine (ETAM). There is a need for compounds that are similar in activity to NTZA, but have greater oral pharmacokinetics and metabolism to release tizoxanide into the bloodstream. A pharmacokinetic (PK) study performed in Sprague-Dawley rats showed that RM5071 was more bioavailable than NTZA in terms of maximum concentration. For the RM5071, it will be necessary to develop a synthetic process capable of upscaling.

1. consideration

1.1. First approach

RM5071 was originally prepared by the following protocol (see also Appendix 1):

Two millimoles of tizoxanide was suspended in 20 mL of methanol containing 0.15 mL of ethanolamine. The suspension was warmed to +50° C. over a few minutes, filtered and the filtrate was concentrated to 5 mL. Crystallization started readily and diethyl ether (5 mL) was added and the mixture was cooled to 0° C. before filtering. The cake was washed with diethyl ether containing a small amount of methanol. After drying, RM5071 is obtained as a yellow crystalline solid (0.49 g).

Nuclear magnetic resonance NMR (NMR 1H and 13C), elemental composition, and electrospray ionization mass spectrometry (MS-ESI negative) confirmed the expected structure. A melting point of between 158°C and 160°C (decomposition) was determined.

This batch of RM5071 was used initially for toxicology/PK studies and later as a reference for process development.

1.2. Developing scalable methods

Development of drug substance (DS) synthesis was carried out in three steps. Starting with the initial synthesis, several laboratory scale tests were performed. Those studies led to a scalable method, which was tested on a larger scale in pilot laboratory trials. A technical prototype batch was prepared as no problems were reported during the pilot lab testing. The intent of this larger batch was to see if the method could be effectively implemented using current manufacturing equipment.

Criteria for scaling up the synthesis of RM5071 included the following:
・Yield ・Time ・Safety ・Purity (e.g. ratio of ETAM/TIZ)
may include one or more of

Constraints may include working temperature (between -5°C and +80°C)

1.2.1. Theoretical Considerations and Synthetic Strategies

RM5071 is the ethanolamine salt of tizoxanide (TIZ). The alcohol moiety (phenol) from TIZ is slightly acidic. Taking advantage of this feature, TIZ is combined with an alkaline molecule, 2-ethanolamine (ETAM), to form an organic salt. Although ethanolamine is not a strong base (compared to other organic bases) from a reactivity point of view, it may be strong enough to interact with the slightly acidic phenol groups of TIZ. The salt can thus be formed by mixing both molecules.

Because of this strong interaction between ethanolamine and tizoxanide, the chemical properties of RM5071 differ from those of tizoxanide. Therefore, a change in the melting point (decomposition) as well as a change in the FTIR spectrum were observed. Such differences are likely associated with intramolecular configuration.

The synthetic scheme for RM5071 can be thought of as a two-step synthesis starting with nitazoxanide (NTZA), the second step being salt formation to produce RM5071.

1.2.2. Small-scale testing

1.2.2.1 Step 1

The preparation of tizoxanide from NTZA has already been investigated previously. One protocol of the method could be:

Dispersing NTZA in 10 Veq of HCl 37% at RT forms a very viscous yellow suspension. The mixture is heated to +50° C. for 24 hours (until complete conversion). The slurry becomes less viscous and easier to stir. After it was cooled to RT, it was diluted twice with water (10 Veq) and filtered. The cake is washed with copious amounts of water followed by methanol. Drying the yellow cake under vacuum gives a good yield of pure tizoxanide.

This method is not easy to scale up as it can have two problems. First, this method utilizes concentrated HCl. This is best avoided on large scales for safety reasons. Second, the method can require large reactors. since a total of 20 Veq is required after dilution with water. This would mean that a 6000L reactor could use up to 300 kg of NTZA in one batch.

During previous projects, attempts were made to react in an organic medium, ie dissolving NTZA in THF and reacting it with aqueous ammonia. The reaction was complete in minutes and no heating was required. However, the workup process (concentration to dryness (evaporation of ammonia), reslurry in water, acidification with HCl, followed by filtration) was not easy to scale up. Several variations of the method were tested; however, this method was abandoned.

The focus was on hydrolysis with HCl. It soon turns out that no improvement is possible in a purely aqueous medium. Thus, working with lower concentrations of HCl results in longer reaction times even at elevated temperatures, and working with lower dilutions results in unstirrable mixtures.

In parallel, the reaction in DMF was also investigated. This is because NTZA is soluble in DMF and slowly degrades to tizoxanide. But even when the temperature was increased and water was added in excess as a catalyst, the reaction rate was really slow. Conversely, addition of substantial amounts of aqueous HCl and heating results in reasonable conversion rates. A brief description of this protocol is provided below:

NTZA is dispersed in DMF (3 Veq) at RT, aqueous HCl (2 Veq 1M) is added and heated to +70° C. until complete conversion. Filtration and washing (water then methanol) gives a good yield after drying.

NTZA is nearly soluble in DMF at working concentrations and at room temperature; however, upon addition of aqueous media, the mixture becomes very difficult to stir until the internal temperature is about +50°C. The stirrability of this mixture increased when NTZA was converted to tizoxanide. Finally, the method was modified by heating the starting solution to +50° C. before adding the acid. No stirring problems were observed during the HCl addition during the reaction.

Solid tizoxanide was recovered after an additional neutralization step by filtering in the laboratory but centrifuging in the plant to avoid handling acidic mixtures in the plant and to prevent centrifugal corrosion. An equal amount of NaOH 1M (aqueous) can be added to the mixture after completion of the reaction to neutralize free HCl in the reaction mixture. However, the reaction produces 1 equivalent of acetic acid, so the reaction mixture remains acidic.

The resulting slurry filtered without problems; filtration was easily accomplished with a sintered glass funnel. The cake was washed with water (3x2 Veq) and methanol (3x2 Veq). Combining the neutralization step with the aqueous wash leaves no HCl in the cake. Avoiding residual HCl in the product can be important. because it may react with ethanolamine during step 2. Washing the cake with methanol allows water to be removed. However, NTZA and tizoxanide are rather sparingly soluble in water as well as in methanol. This way, tizoxanide is not lost during cake washing, but residual NTZA is also not removed.

In conclusion, a scalable method for preparing tizoxanide from NTZA was obtained.

Disperse NTZA in 3 Veq of DMF at RT. Heat to +50° C. and add 2 Veq of HCl 1M (aq.). Then heat to +70° C. until complete conversion. Cool to RT, add 2 Veq NaOH 1 M (aq.), filter and wash the cake with 3 x 2 Veq water and 3 x 2 Veq methanol. Drying the cake under vacuum gives tizoxanide as an off-white powder.

Good yield and good purity (100% UV range by HPLC). The method is robust, but the response time is different and varies between 16 and 40 hours. The temperature of the reaction mixture can be important for the conversion rate. Reaction times are longer below +70°C (internal temperature). Particular attention should be paid to this during pilot laboratory scale-up. Another consideration is the stirrability and filterability of the reaction mixture. because they are known parameters that scale-up can affect.

1.2.2.2 Step 2

This previous method was reproduced as a starting point for method optimization. This previous method gives the product in good purity but in low yield (36%). Some steps from this method, namely hot filtration, distillation, use of diethyl ether, as well as large dilution (working concentration 0.1 M or 38 Veq) are not suitable for large-scale production. The heating and filtering steps may not be necessary unless some free tizoxanide remains in the suspension, but given the high dilution it is unlikely. A new protocol was therefore designed to avoid the heating and filtration steps as well as the steps of concentration and co-solvent crystallization.

Tizoxanide was dispersed in an organic solvent, ethanolamine was added and the resulting slurry was filtered and washed with solvent. Several tests have shown that methanol is an excellent solvent for the process and can be used at a working concentration of 0.75 M (or 5 Veq). However, since the final product is slightly soluble in methanol, it was found that washing the cake with a mixture of methanol and ethyl acetate (1/1 v/v) gave good results.

RM5071 is insoluble in ethyl acetate but tends to form a sticky material. This was also observed during test reactions in pure ethyl acetate. In addition, RM5071 was observed to be nearly soluble in methanol at a concentration of 5 mg/mL.

Time was also spent analyzing the final material during the development of Step 2 of the synthesis. The organic salt as final material consists of two starting materials from the process, which may not be as easy to analyze as in classical organic reactions. This topic will be discussed later.

No need for purification was observed, but it was studied nonetheless. Two methods were tested: slurry and dissolution/crystallization.

Slurries were investigated in water and methanol. Because both are expected to remove ethanolamine.HCl salts as well as possible traces of free ethanolamine. Residual tizoxanide could not be removed in this way. Both solvents showed increased purity (ETAM/TIZ ratio) and good yields:

Dissolution/crystallization was retested in DMF but not in methanol:

RM5071 was dissolved in a minimal amount of DMF (2.5 Veq) at RT. The final solids can be filtered, but no solids are observed. Addition of 25 Veq of ethyl acetate can precipitate RM5071 at RT. After filtration, the product is washed with methanol/ethyl acetate (1/1).

The purification rate was about 70%. Excess ethanolamine is removed, otherwise no gain is observed compared to other purification methods or even no purification.

The reaction was also worked out in DMF and the product was recovered by co-crystallization with ethyl acetate. Since crystallization requires analysis of the final material relative to other batches of high dilution, this method may show no advantage.

In conclusion, the methanol suspension reaction followed by simple filtration was kept in step 2:

Tizoxanide is dispersed in methanol (5 Veq) at RT. Addition of ethanolamine (1.1 eq) limits the exotherm. After stirring the mixture for 2 hours, it is filtered and washed with a mixture of methanol/ethyl acetate (1/1 V/V).

Yield is about 80%. Analysis shows good purity, with an ETAM/TIZ ratio between 0.90 and 1.00.

If the final material contains excess ethanolamine or another salt, it can be purified by reslurrying it in methanol and/or water.

A higher excess of ethanolamine was also tried since free tizoxanide was detected in the final material. No difference was observed in the final material. One non-limiting hypothesis would be that traces of ethanolamine may have dissociated from tizoxanide during the washing step (filtration) resulting in this slight excess of tizoxanide.

1.2.2.3 Conclusion

A two-step process from NTZA to RM5071 capable of upscaling was developed.

Step 1: Preparation of Tizoxanide from NTZA

After dispersing nitazoxanide in 3 Veq of DMF, the mixture is heated to +50°C. Slowly add HCl 1M (aq, 2 Veq). The mixture turns from a yellow solution to a white solution. It is heated to +70° C. until conversion is complete (generally 36-48 hours).

The mixture is cooled to RT and NaOH 1M (aq, 2 Veq) is added slowly to neutralize the HCl. Temperature would need to be controlled and cooling would need to be extensive.

The suspension is filtered and the solid is washed 3 times with 2 Veq water and then 3 times with 2 Veq methanol. The solid is dried under vacuum to give pure tizoxanide.

Step 2: Preparation of RM5071 from Tizoxanide

Disperse tizoxanide in 5 Veq of methanol at RT. Ethanolamine (1.1 eq) is slowly added while maintaining the internal temperature below +30°C. Cooling may need to be extensive. The suspension changed from grayish white to yellow. The mixture is stirred for 2 hours at RT, then filtered and the solid is washed 3 times with 2 Veq of a mixture of methanol and ethyl acetate (1/1 V/V). The yellow powder is dried under vacuum (oven).

Grinding the solid gives a fine yellow powder.

met the criteria for upscaling:
Yield: 80-90% in 2 steps
time:
Step 1: Reaction time can be 20-46 hours of heating. This parameter could be further improved.
Step 2: Reaction time can be set to 2 hours, but chemically the formation of RM5071 is instantaneous Safety Do not use concentrated acids/bases Do not handle strongly acidic or alkaline mixtures Some exotherm to control Volatility Solvent-free Purity
HPLC-UV, HPLC-MS showed good purity
qNMR showed an acceptable ratio ETAM/TIZ Particle size distribution appeared excellent Working temperature (between -5°C and +80°C)
Cooling may be required to maintain temperature near RT while adding some heat
o Heating up to +70°C (internal) may be achieved.

According to this chosen method, the following quantities could be prepared:

1.2.3 Pilot laboratory test

As a second part of method development, selected methods were tested at a pilot laboratory scale. Two batches were prepared: one using classical glassware and one using a glass pilot reactor.

The following are the main conclusions regarding the optimization of the synthetic method:
• Addition of reagents (HCl, NaOH, and ETAM) did not cause problematic temperature increases. However, sufficient cooling may be required when adding NaOH and ETAM (set internal temperature cooling to +10°C).
• The reaction time during step 1 can be about 45 hours. A minimum temperature inside the reactor of +70° C. may be required to achieve this reaction time. Temperatures below this level will result in longer reaction times.
• A 50 µm filter cloth can be used for centrifugal filtration.
• No difficulties were found with agitation (158 rpm, Buchi reactor). The critical part may be when near complete conversion is reached during step 1.
• The product can be easily removed after both steps, leaving little residue. The reactor is easy to clean.
• Carry out step 2 by returning the intermediate (tizoxanide) to the reactor without drying. Determine loss on drying (LOD). After adjusting the amount of methanol depending on the LOD results, it can be loaded into step 2.

Yield was 88% on a scale of 1.5 kg starting material and purity was consistent with other development batches. Since no critical parameters needed to be modified, it was decided to test the method directly on a larger scale (50 kg starting material).

1.2.4. Engineering Batch

The main purpose of the engineering batch was to investigate the suitability of this method for manufacturing RM5071. Parameters to be checked were the ability to heat the step 1 reaction mixture to an efficient temperature (+75°C internal) and the ability to recover tizoxanide (step 1) and RM5071 (step 2) during centrifugation. Ta.

Conclusions regarding synthetic development are as follows: Yield: 78% over two steps.
·time:
• Step 1: 20 hours heating; 32 hours total production time.
• Step 2: 2 hours reaction time; 7 hours total production time.
Safety Exothermic reaction easily controlled Purity No difference observed compared to previous batches Working temperature (between -5°C and +80°C)
・Cooling power was sufficient. ・Heat of +75°C was achieved. Sufficient heating power was utilized.

In conclusion, 41 kg of RM5071 drug substance was prepared as an engineering batch according to the synthetic method developed at pilot laboratory scale.

1.3. Analysis Considerations

Analysis of the final RM5071 was difficult. RM5071 is an organic salt consisting of tizoxanide and ethanolamine. Therefore, classical analytical techniques cannot be used directly for final product evaluation. A method is needed that can distinguish the free reagents (ETAM and TIZ) from the final product (RM5071). During synthesis development, we chose to reveal the ratio ETAM/TIZ for that purpose:

This molar value should be 1.00 according to the chemical structure of RM5071. If free tizoxanide or ethanolamine were present in the final product, the observed ratio would change, as shown in 5, allowing quantification of residual starting material. Other organic impurities are easier to identify (HPLC, NMR). No inorganic impurities are expected in the final product.

It was also important to analyze the intermediate (tizoxanide) in addition to the final product, both to monitor the conversion during step 1 and to start the final step with pure material. The LC-MS method allows for highly reliable results, rapid results, and easy sample preparation.

During synthetic development, one important parameter for the thiazolides, the size distribution of the particles was also checked by laser diffraction to ensure that changes in method did not affect this distribution.

2. Conclusion

In conclusion, the following synthetic protocol can be utilized to prepare RM5071 drug substance from laboratory scale to manufacturing scale:

Preparation of tizoxanide from NTZA (Step 1)

After dispersing tizoxanide in 3Veq DMF, the mixture is heated to +50°C. Slowly add HCl 1M (aq, 2 Veq). The mixture turns from a yellow solution to a white suspension. It is heated to +75° C. until conversion is complete.

After cooling the mixture to +10/+15°C, NaOH 1M (aqueous, 2 Veq) is added slowly to neutralize the HCl while maintaining the internal temperature below +25°C.

The suspension is centrifuged (or filtered on a small scale) on a 50 μm filter cloth and the solid is washed 3 times with 2 Veq water and then 3 times with 2 Veq methanol. The solid is used directly in the next step without further drying.

Preparation of RM5071 from Tizoxanide (Step 2)

Disperse tizoxanide in 5 Veq of methanol at RT. Cool the mixture to +10/+15°C. Ethanolamine (1.1 eq) is slowly added while maintaining the internal temperature below +25°C. The suspension turned from off-white to yellow. After stirring for 2 h at RT, the mixture was centrifuged on a 50 µm filter cloth (filtered on a laboratory scale) and the solid was removed with 2 Veq of a mixture of methanol and ethyl acetate (1/1 V/V). Wash 3 times with The yellow powder is dried under vacuum (+50°C, <100 mbar). Grinding the solid if necessary gives RM5071 as a fine yellow powder.

Appendix 1: Original Protocol RM5071

2-Hydroxybenzoyl-N-[(5-nitro)thiazol-2-yl]amide, ethanol salt

Tizoxanide (i.e., 2-hydroxybenzoyl-N-[(5-nitro)thiazol-2-yl]amide, 0.53 g, 2 mmol) was suspended in methanol (MeOH, 20 ml) containing ethanolamine (0.15 mL). made it cloudy. The suspension was warmed to 50° C. over a few minutes to give a virtually clear yellow solution that readily began to crystallize when filtered and concentrated to 5 mL. Diethyl ether (Et ₂ O, 5 mL) is added and the mixture is cooled to 0° C. for complete crystallization. Filtration and washing with Et ₂ O containing a small amount of MeOH gave the title salt as a yellow crystalline solid (0.49 g, 75%); mp 158-160° C. (decomposition); H, 4.2; N, 17.35; S, 9.8. _{C12H14N4O5S requires} C, _44.2 ; H _, 4.3; N, _17.2 ; _S , 9.8%; _{2CH2 )} , 3.57 (2H, t, _CH2CH2 ) _, 5.20 (1H, brs, OH), 6.81 (2H, m, ArH), 7.32 (1H, m, ArH), 7.67 (3H, br s, _NH3 ⁺ ), 7.91 (1H, m, ArH), 8.51 (1H, s, 4'-H), and 14.71 (1H, brs, NH); C NMR [100 MHz, ( _CD3 ) ₂ SO] 41.6, 57.9, 117.5, 118.2, 119.9, 130.1, 133.4, 137.9, 145.9, 161.3, 171.6, and 172.2. ; m/z (-ve ion electrospray mode) 264 [(MH) ^? ]. Measured value: m/z, 264.0092. _C10H6N3O4S claims _{m /} z, _264.0085 .

Example 14

RM5071 is a prodrug of tizoxanide (TIZ, also known as desacetyl-nitazoxanide or desacetyl-NTZA), an active metabolite of nitazoxanide (NTZA) and is approved by the FDA for children (oral suspension) and adults ( An antiprotozoal drug (Alinia) approved for the treatment of Cristopolidium parvum or Giardia lamblia in tablets).

RM5071 is an organic salt consisting of two ionic moieties, tizoxanide alkoxide and ethanolammonium. The chemical structure of ethanolammonium tizoxanide alkoxide (hereafter referred to as RM-5071) is shown below. The lab-scale synthesis of RM-5071 is essentially a two-step synthesis using NTZA as the starting material. The first step is to remove the acetyl group of NTZA by dissolving it in warm (70 °C) dimethylformamide (DMF) in the presence of hydrochloric acid (HCl), followed by neutralization with sodium hydroxide (NaOH) at room temperature. It is to do. The product obtained is filtered before the next step. The second step involves dispersing the filtered powder in methanol and then cooling the suspension while slowly adding ethanolamine to form the salt. The solid is filtered, washed and dried in the Rotavapor. The final product is a yellow powder identified as RM-5071.

Chemical structure of RM5071 (hydroxyethylammonium tizoxanide alkoxide).

This report summarizes the physical and chemical characterization data obtained to date for the above synthetic products. The purpose of physical characterization is to obtain information on characteristics such as melting point, particle size distribution, crystallinity, morphology, and performance under thermal stimulation. Chemical characterization data provide information on solubility, acid-base properties, chemical function, molecular weight, and spectral features to gain knowledge about the chemical behavior of molecules.

1. consideration

This section describes performed analytical tests, data, and results from various physical and chemical characterization techniques listed below:
Physical characterization Visual inspection Particle morphology by scanning electron microscopy (SEM) Particle size analysis by laser diffraction Calorimetric transitions monitored by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) Characterization Solubility Acid-base titration UV-Vis spectroscopy (UV)
・Proton nuclear magnetic resonance ( ¹ H-NMR)
・Electrospray-mass spectrometry (ESI-MS)
・Fourier transform-infrared spectroscopy (FT-IR)
・X-ray diffraction (XRD)

A sample of RM-5071 and a sample of desacetyl-NTZA (tizoxanide) were submitted for comparison purposes.

1.1. Physical Characterization

1.1.1. Visual inspection

Visual inspection of RM-5071 appears as a light yellow powder containing fine particles agglomerated into easily breakable clumps. By comparison, the desacetyl-NTZA sample appears as a discrete, bone-white powder containing fine particles.

1.1.2. Scanning electron microscopy

Scanning electron microscopy (SEM) is an analytical technique used to obtain magnified images of sample morphology. This is achieved by focusing the electron beam on the sample and controlling the accelerating voltage and thus the penetration depth and kinetic energy of the incident electrons to acquire both backscattered and secondary electron signals. An energy dispersive spectroscopy (EDS) system is utilized in combination with an SEM system to obtain the elemental composition of the sample.

1.1.2.1 Materials and Equipment

The instrument used for SEM/EDS analysis by MCC was a JEOL 6480 LV scanning electron microscope equipped with an EDAX X-ray fluorescence detection unit.

1.1.2.2 Procedure

This microscopy was performed by the Material Characterization Center (MCC). Two samples were submitted to the Materials Characterization Center (MCC), San Juan, Puerto Rico for characterization and comparison. The material was separated with the aid of a spatula and secured to double-sided carbon tape that had been pre-glued to a piece of aluminum. Backscattered electron images (BEI micrographs) were acquired at 1500x magnification. Particle size analysis was performed by coating the samples with a thin film of gold approximately 30 nm. Both analyzes were performed at 20 kV in high vacuum. Secondary electron images (SEI) of the samples were acquired between 500x and 1000x magnification. Figures 2 and 3 show electron microscope images at two different magnifications for both RM 5071 and desacetyl-NTZA. As shown in Figure 6, the RM-5071 particles exhibit a morphology that appears to be formed by layers and/or steps. Most of the single particle shapes are elongated; nevertheless, aggregates containing irregularly shaped particles and rounded-edge particles can be seen in both regions examined. Conversely, the desacetyl-NTZA SEM image in Figure 7 shows elongated particles with sharp edges.

A visual comparison of SEM images from RM-5071 (Figure 6) and desacetyl-NTZA (Figure 7) at the same magnification reveals that RM-5071 contains slightly smaller particles than desacetyl-NTZA. looks like it does. In addition, a preliminary analysis of particle size was performed in MCC using information from SEM images by measuring samples of 10 particles per spot. The RM-5071 samples ranged in particle size from approximately 4.16 to 26.30 μm with an average of 13 μm. The particle size of desacetyl-NTZA was approximately 3.45-34.30 μm with an average of 15.1 μm. These results are shown in Table 5. In addition, the EDS results showed that the elemental composition was similar for both RM-5071 and desacetyl-NTZA: carbon as the major element; oxygen and sulfur as minor elements; and nitrogen as the minor element. Additionally, traces of aluminum were present on both the RM-5071 and desacetyl-NTZA samples due to the piece of aluminum used to hold the carbon tape onto the sample stage for introducing the sample into the microscope. Existing.

1.1.3. Analysis of particle size distribution

Laser diffraction is used as a particle size measurement method in the range of 0.5-1000 microns. It is based on the principle that when a light beam (laser) is scattered by a group of particles, the angle of light scattering is inversely proportional to the particle size.

1.1.3.1. Methods

Guidelines for suitable methods for particle size distribution (PSD) on RM-5071 samples using laser diffraction are provided in ISO 13320-2009: Particle Size Analysis - Laser Diffraction Method and USP <429> Particles. Developed on the basis of optical diffraction measurements of size.

1.1.3.2. Materials and Equipment

The instrument used for particle size distribution analysis was a Malvern Mastersizer 3000. Dry dispersion was carried out using a venturi-type disperser. For liquid dispersion, prepare a particle analysis sample by dispersing the solid in IPG (Isopar G, an isoparaffinic hydrocarbon) containing 2% lecithin and sonicating for 15 seconds using an Elmasonic S ultrasonic bath. It was implemented by

1.1.3.3. Procedure

The purpose of Phase 1 of method development is to evaluate sample preparation conditions and instrument settings for particle size analysis of RM-5071 by laser diffraction.

1.1.3.4. Results

The RM-5071 sample was first examined by light microscopy to reveal the general particle size and shape before proceeding to evaluation. The particles were observed to be irregularly shaped, with primary particles typically no larger than 40 μm. Soft to semi-rigid agglomerates are found in powders in the millimeter size range. These agglomerates can be dispersed fairly easily by pressure (for example pressing the agglomerates with the tip of a spatula).

Using default settings and varying dispersants and bases, analysis of the size distribution of liquid dispersed particles was performed to reveal suitable dispersions. Some dispersion energy was required to fully disperse the sample material into primary particles. Preparations were sonicated for 15 seconds using default settings before analysis. Figure 8 shows a digital image of the sample preparation dispersion before and after 15 seconds of sonication, aggregates are dispersed after sonication. The formulation using IPG (Isopar G, an isoparaffinic hydrocarbon) with 2% lecithin as the base and dispersant appeared to produce the most homogeneous dispersion by microscopic observation. These conditions were utilized in comparative analysis with dry ANOVA.

To analyze the size distribution of the dry-dispersed particles, pressure titration was performed to assess the effect of pressure on the RM-5071 particles. The particle size results from this analysis are summarized in Table 3. Note that the results of PSD analysis by laser diffraction confirm the preliminary observations by SEM that the individual particles have a rough size of 3-40 μm and aggregates of primary particles are also observed. .

A superposition of the distributions produced at each pressure is shown in FIG. 9 compared to the average liquid distribution. The pressure that produces results most similar to wet dispersion (indicated in red) is the optimal pressure to choose for analysis, which is 1 bar as indicated by the blue line in Figure 9. is. The particle size distribution results reveal that the aggregates present in the sample were not sufficiently dispersed by the attempted dry dispersion setup. The reason is that a small population of coarse particles with a size of 100-1200 μm is observed. Furthermore, as the pressure increases, the main peak of the distribution shifts to smaller particle sizes as expected. This is because higher pressures can destroy primary particles. At the highest pressure (4 bar) there is still a small amount of coarse particles present in the dry dispersion analysis. This indicates that small amounts of aggregates are still observed even at this higher energy setting.

The feed rate and hopper height settings were initially chosen to allow a steady and complete flow of the sample, but at the end of the pressure titration analysis, the settings initially chosen were sufficient to keep the powder constant. It turned out to be insufficient to feed the device. Instrument settings were adjusted to improve sample flow, as non-ideal sample flow may have contributed to incomplete dispersion of aggregates. Pressure titration analyzing at 4 bar with new feed rate (65%) and hopper height (1.5 mm), particle size distribution remained similar despite improved sample flow.

To aid dispersion, aggregates in the aliquot to be analyzed were dispersed using a spatula prior to addition to the instrument. This worked so that the coarse particle peak was not present in the analysis (Figure 10), but the peak shifts to smaller particle sizes compared to the liquid analysis. Based on these analyses, preliminary factors for method development were selected as shown in Table 4. This will suffice to characterize the material at this time, although it will require further method development to be optimized.

1.1.4. Melting point

The material exhibits a melting transition from the temperature at which a first detectable change in liquid phase is detected to the temperature at which the solid phase disappears. This transition can appear instantaneous in very pure materials, but is usually observed over the course of the process. Factors influencing this transition can include, among other variables, sample size, particle size, efficiency of heat diffusion within the sample, and heating rate, which are controlled by procedural instructions. be done.

1.1.4.1. Methods

Melting points were determined according to standard operating procedure BEL-SOP-000152 entitled "Melting Point Apparatus BUCHI B-540".

1.1.4.2. Materials and Equipment

The instrument used for melting point analysis was a Buchi B-540.

1.1.4.3. Procedure

A volume of RM-5071 and desacetyl-NTZA corresponding to a height of 4-6 mm in the melting point capillary was inserted into the Buchi B-540.

1.1.4.4. Results

Differences in melting points of structurally similar organic molecules are useful for distinguishing properties. Both RM-5071 and desacetyl-NTZA are solids at room temperature. Melting points of RM-5071 and desacetyl-NTZA were determined. The results are shown in the table.

These results show that the melting temperature of RM-5071 (146-148°C) differs from that of its precursor desacetyl-NTZA (240-250°C). This difference is a distinguishing factor for the useful physical properties of RM-5071 as a new chemical entity. It provides a means of distinguishing RM-5071 from desacetyl-NTZA during the synthetic process.

1.1.5. Loss on drying

Loss on drying (LOD) is a test method that determines the moisture content of a sample, but can optionally refer to the loss of any volatile material from the sample.

1.1.5.1. Materials and Equipment

LOD measurements were performed in a non-certified apparatus and heating was performed in a Binder vacuum furnace with a vacuum pump Vacuumubrand MD4C (Pmin = 1.5 mbar) with an accuracy of ±0.1°C. The mass of the sample was determined using an analytical balance Mettler AG285 (d=0.01 mg).

1.1.5.2. Procedure

After the RM-5071 sample was lightly pressed with a spatula to break up any agglomerated particles, the test sample was weighed. A sample was placed in a weighing bottle and its mass was measured by the difference before and after heating to 60° C. for 2 hours in a vacuum oven. Drying was continued until two consecutive weighings differed by less than 0.50 mg/g of material taken, and drying was continued for an additional hour after the second weighing. This procedure is performed in accordance with USP <741> loss on drying.

1.1.5.3. Results

LOD measurement of RM-5071 by USP method is 0.2%. The sample weight remained stable after the second drying round. A low percentage of loss on drying suggests that volatiles that may have been left over from the synthetic process are not adsorbed onto the solid RM-5071. This result is consistent with the data obtained from thermogravimetric analysis (TGA) discussed in the next section, where RM-5071 shows no significant weight loss up to 100°C.

1.1.6. Calorimetric Analysis

Differential scanning calorimetry (DSC) is one thermal analysis technique that measures the difference in the amount of heat required to raise the temperature of a sample relative to a reference as a function of temperature. The reference sample should have a well defined heat capacity in the temperature range to be scanned. One application of DSC is the study of phase transitions (such as melting, glass transition, and/or exothermic decomposition). Such measurements provide qualitative and quantitative information about physical and chemical changes in molecules.

Thermogravimetric analysis (TGA) is an analytical technique used to determine the thermal stability and volatile content of materials by monitoring the weight change that occurs when the material is heated. Since many weight loss curves look similar, they may have to be transformed before the results can be interpreted. A differential weight loss curve can be used to identify the point of most pronounced weight loss. Analyzes are usually performed in air or in an inert atmosphere (such as nitrogen).

1.1.6.1. Materials and Equipment

The instruments used for DSC and TGA were the TA Instruments DSC Q2000 and TA TGA Q500 instruments equipped with the TA universal Analysis 2000 program, respectively.

1.1.6.2. Procedure

Calorimetric analysis was performed by MCC. For DSC analysis, 2.34-2.46 mg of sample RM-5071 and desacetyl-NTZA were individually placed in aluminum airtight pans and covered with aluminum lids. The temperature method was step 1: hold at 25°C for 10 minutes and step 2: heat from 25°C to 400°C at 10.00°C/min. Additional DSC experiments were performed to clarify further crystallization behavior by performing heat-cool-heat cycling experiments on RM-5071. Approximately 1.69 mg of RM-5071 sample was placed in an aluminum airtight pan and covered with an aluminum lid. The temperature method is step 1: hold at 25°C for 5 minutes, step 2: heat from 25 to 350°C at 10.00°C/min, step 3: cool from 350 to 25°C at 10.00°C/min; : Heating at 10.00°C/min from 25 to 350°C. Both analyzes were performed under nitrogen.

For TGA analysis, 2.0-2.6 mg amounts of both RM-5071 and desacetyl-NTZA samples were individually placed in platinum pans. The temperature method was step 1: equilibrate at 25°C, step 2: heat from 25°C to 700°C at 10.00°C/min, step 3: hold at 700°C for 5 minutes. Analyzes were performed under nitrogen.

1.1.6.3. Results

Figure 11A (left) contains the thermogram of RM-5071 with a first stage weight loss of approximately 20% in the temperature range of 85-160°C and total weight after three separate thermal transitions. Indicates a loss of 77%. The initial weight loss of about 20% is consistent with the hypothesis that the loss is ethanolammonium (average molecular weight = 62.09 amu), 19.03% of the total weight of RM-5071 (average molecular weight = 326.33 amu). Desacetyl-NTZA had a first stage weight loss of 36% in the temperature range 199-280°C and a total weight loss of about 67% after two transitions. Based on these results, the thermal properties of RM-5071 and desacetyl-NTZA are significantly different, especially with respect to the number and temperature of thermal transitions undergone by both molecules. The presence of ethanolammonium thus results in different thermal stability behavior between the desacetyl-NTZA precursor and its ethanolammonium salt.

The DSC of RM-5071 in Figure 12 shows a single peak transition at 163°C. This transition temperature is lower than the 286° C. transition temperature observed in the DSC thermogram for desacetyl-NTZA. Both molecules exhibit an exothermic transition. Results of heat-cool-heat cycling experiments on RM-5071 indicate that the 163°C transition is irreversible and may be attributed to ethanolammonium loss and/or molecular decomposition. ing. In conclusion, the precursor desacetyl-NTZA is more thermally stable than its ethanolammonium salt.

1.2. Chemical characterization

1.2.1. Solubility

According to the USP, solubility is the ability of a solvent to dissolve a solute and is defined in units of concentration. Solubility of solids is a function of polarity and temperature. Apparent solubility is the empirically determined solubility of a solute in a solvent, allowing insufficient time for the system to reach equilibrium. Equilibrium solubility, on the other hand, is the solubility limit at thermodynamic equilibrium toward which a solute can dissolve uniformly in a solvent when an excess of solid is present. Understanding the solubility of RM-5071 may be important for developing validated analytical methods for quantifying analytes and their related substances. In addition, the knowledge derived from solubility studies is the basis for manufacturing the final drug product.

1.2.1.1. Methods

The apparent solubility of RM-5071 was investigated using several solvents, specifically DMF, DMSO, acetonitrile (ACN), water, ACN/water mixtures, methanol, isopropanol, and ethanol. The United States Pharmacopeia (USP) classifies solubility simply according to the criteria of definitions summarized in Table 10, regardless of the solvent used. Solutes were initially dissolved in their respective solvents or solvent systems at a concentration of 1 mg/mL. If the solute remained undissolved, then a new solution of lower concentration was prepared until complete dissolution of the solid was observed. This concentration limit was taken as the apparent solubility.

1.2.1.2. Materials and Equipment

All chemicals used in the solubility tests are specified in Table 11.

1.2.1.3. Procedure

A solution of RM-5071 was prepared by dissolving 10 mg of RM-5071 in each solvent while increasing the volume of the solvent from 10 mL to 100 mL and then to 1000 mL. Following visual inspection and observation, the apparent solubility of RM-5071 was classified according to its 10 as defined by USP.

1.2.1.4. Results

Solubility studies of RM-5071 were conducted at room temperature in several solvents specified in Table 11. For the purposes of this chemical characterization report, the terms specified in Table 10 were used to describe the solubility of RM 5071 in different solvents in Table 12.

RM-5071 was not completely soluble in water at concentrations as low as 0.005 mg/mL. Lower concentrations were not tried due to practical limitations (ability to weigh less than 10 mg and unavailability of flasks larger than 1 L). Some amount of solute is still soluble in water, as evidenced by the yellow color of the solution and observation of the UV-vis spectrum in the supernatant. This is further discussed in Section 5.2.3. The RM-5071 solution prepared at a concentration of 1 mg/mL was bright yellow and turned orange over time. This may indicate that the molecule may degrade or be unstable in water.

RM-5071 is highly soluble (>1 mg/mL) in ACN:water (65:35) mixtures. This finding is somewhat surprising. This is because the solubility in pure acetonitrile and water is much lower (≤0.033 mg/mL and <0.005 mg/mL, respectively). However, according to Qiu, et al. Organic Process Research & Development 2019, 23 (7), 1343-1351, for several charged solutes (APIs) investigated, mixing an organic solvent with a small amount of water resulted in increases relative to the solubility of the solute in the respective pure solvent. This behavior is known as a synergistic solvation effect (also known as parabolic solubility). The same effect was observed with RM-5071 in 25:75 and 50:50 water:alcohol (isopropanol and ethanol) mixtures.

1.2.2. Acid-base titration curve

Direct titration involves treating a soluble substance (the titrant) in solution (the titrant) in a suitable container with a suitable standardized solution (titrant) and measuring the appropriate It is to reveal the endpoint of the reaction with the aid of indicators. For acid-base titrations, when plotting a series of pH measurements as a function of the volume (mL) of titrant added to the titrant, a sigmoidal curve with a sharp change near the equilibrium point is obtained. can get. Chemical information such as pKa (acid/base equilibrium constant) can be derived from such curves.

1.2.2.1 Materials and Equipment

The pH meter used for measurements during titrant addition was a Mettler-Toledo Seven Excellence. Table 13 contains all materials used during the experiments.

1.2.2.2. Procedure

Acid titration was performed by pouring 20 mL of a 1 mg/mL RM-5071 solution (substance to be titrant) into a beaker and measuring the pH of the solution while using 0.01N HCl solution as the titrant. did. Alkaline titration was performed by adding 20 mL pours of 1 mg/mL RM-5071 solution to a beaker and measuring the pH of the solution while adding 0.02 N NaOH solution as titrant. The solution was stirred with a magnetic stirrer during the titration and stopped during the pH measurement.

1.2.2.3. Results

A change in color and solubility of the 20 mL RM-5071 solution was observed as the acid titration proceeded by adding 0.01 N HCl to the RM-5071 solution. Initially, a 1 mg/mL RM-5071 solution has a pH of 9.44. As acid is added to the beaker and titrated, the pH drops as expected. In addition, as the pH decreases, the solution becomes increasingly cloudy and solids precipitate. At a pH of 3.80, the physical appearance of the contents of the beaker is a beige powder suspended in a pale yellow solution. The appearance of the suspended precipitated powder is similar to desacetyl-NTZA. This suggests that the hydroxyl group of the phenol in the molecule was protonated, thus reducing its polarity and consequently its solubility in water and precipitating out of solution.

The titration curve in Figure 13 shows the inflection point for the acid-base titration of RM-5071. The inflection point was calculated using the second derivative of the experimental curve, the equivalence point volume is 5.83 mL of 0.01 N HCl at pH=5.99. Using this information and the mass of RM-5071, the calculated stoichiometry of the acid-base reaction resulted in one proton per RM-5071 molecule. The half-volume point of inflection is 2.92 mL, giving a pKa of 8.8 for RM-5071. The proton pKa of phenol is 9.9 (Vollhardt, KPC; Schore, NE, Organic Chemistry; Palgrave Version: Structure and Function. Macmillan International Higher Education: 2014). Therefore, the proton titrated with an acid with a pKa estimate of 8.8 is the circled proton of the phenol below:
It is reasonable to conclude that

An image of the beaker containing the RM-5071 solution as a 0.01 N NaOH solution showed the solution gradually changing from a cloudy yellow suspension to a clear orange with increasing pH. It is observed that more and more RM-5071 dissolves with increasing pH and complete dissolution of the solid is achieved at pH=10.63. No pKa information can be derived from this titration curve in the acidic pH range, as the titration curve is in Figure 14 and no clear inflection point can be found. One possible, but non-exclusive, explanation for the absence of a distinct inflection point in the titration curve is that the RM-5071 molecule can undergo various resonance species after deprotonation of the amide nitrogen. There is a possibility. Such an explanation may be consistent with the observed absorption of solutions at higher wavelengths (redshift).

1.2.3. UV-Vis spectroscopy

The wavelength of absorption (λ) corresponds to the energy difference between the ground state and the excited state. When molecules absorb UV-visible light energy, electrons move from occupied orbitals to unoccupied orbitals with higher potential energy. An electron can make several possible transitions with different energies. In the case of RM-5071, the most likely transitions are σ→π^* and π→π^*, which are characteristic of chemical functionalities carbonyl and conjugated carbon double bonds, respectively. The transitions at which electromagnetic waves are absorbed in the UV-Vis region of the spectrum are transitions between electron energy levels and are directly related to the wavelength of absorption.

The intensity of absorption is related to the concentration of the species in solution. The Beer-Lambert law defines the relationship between the intensity of visible UV radiation at a particular wavelength (λ) and the concentration of substances present in an analyte. Beer-Lambert law: A=εlc, where A is the absorption at λ, ε is the molar absorption coefficient at λ, l is the path length and c is the concentration of the species. The UV-visible spectroscopy results from the characterization experiments performed have two aspects: electronic absorption spectra in various solvents and quantification of molecules in the supernatant solution based on absorption at λ _max in water. provides chemical information about

1.2.3.1 Materials and Equipment

A list of materials used during UV-Vis spectroscopy is shown in Table 11. The equipment used in this study is the Shimadzu UV-1800 series.

1.2.3.2. Procedure

UV-Vis analysis was performed by preparing a solution of approximately 1.00 mg of RM-5071 in a 1.0 L volumetric flask and filling the solvent to volume. The resulting solution (supernatant if solids were still present) was transferred to a quartz cuvette with a standard path length of 10 mm. UV-Vis spectra of RM-5071 were performed in the following solvents: DMF, DMSO, water, ACN/water, methanol, isopropanol, and ethanol. Instrument-specific conditions are listed in Table 14.

.2.3.3. Results

The λ _max of RM-5071 in different solvents are summarized in Table 12. Compounds with darker colors (absorbing in the visible region) likely contain long-chain conjugated systems or polyaromatic chromophores. Figure 15 shows representative UV −Vis absorption spectra are shown, with different solvents giving similar spectra but slightly shifted according to the λ _max shown in Table 15. Benzenoid compounds can develop color if they have sufficient conjugated substituents;

A linear relationship between the absorbance at a particular λ and the concentration of the analyte in solution is used to quantify the analyte using the Beer-Lambert law. Aqueous solutions of RM-5071 were prepared at 0.051, 0.025, 0.010, 0.0075, and 0.0051 mg/mL. These solutions left small clumps of solids at the bottom of the flask, so sonication was used to help dissolve the solids. After 5 minutes of sonication, solid particles were no longer observed except for the 0.05 mg/ml solution. The absorbance of the supernatants was measured after the solutions were allowed to settle for 1 hour. These solutions were later centrifuged and the spectrum of the supernatant solution was also acquired. UV-Vis absorption was measured at λmax = 409 nm.

Figure 16 contains the relationship between absorbance and theoretical concentration of RM-5071 in water. The red curve represents the absorbance measurements of the supernatant after precipitation and the blue line represents the absorbance measurements of the supernatant after centrifugation. A comparison of these two absorbance versus concentration curves shows that the r2 (correlation coefficient) is closer to 1 for the blue line (0.99) than for the red line (0.97). This indicates that the concentration of RM-5071 reaches equilibrium after centrifugation. Because the linearity of this plot shows that the Beer-Lambert law is met. This therefore indicates that centrifugation helped dissolve the solids.

1.2.4. Nuclear magnetic resonance

Proton nuclear magnetic resonance ( ¹ HNMR) is a technique that can provide chemical structural information of organic molecules. This technique is based on low-energy absorption at radio frequencies (λ=1-5 m) of proton nuclei in strong magnetic fields. The basis of ¹ H NMR is that proton atoms in different chemical environments have slightly different energy levels in the presence of an external magnetic field, resulting in different radio frequency energy absorption in their ¹ H NMR spectra. An NMR spectrum contains information that can be used to derive the functional groups of a molecule based on how the hydrogen atoms are shielded from the surrounding magnetic field. For example, in the ¹ H NMR spectrum it is possible to determine the number of different types of hydrogen nuclei and to obtain information about the nature of the immediate environment surrounding each type. This chemical information is valuable. This is because it can be used in combination with information obtained from other chemical characterization techniques to derive and/or confirm a proposed chemical structure for a given synthetic molecule. In addition, the integration of the areas under the spectral peaks can be used to derive relative quantitative information regarding the molar ratios between the components of the RM-5071 salt.

1.2.4.1. Materials and Equipment

NMR experiments were performed at the Catholic University of Leuven, Belgium (KUL) using a 400 MHz Brucker NMR.

1.2.4.2 Procedure

NMR measurements were performed by dissolving 5 mg of sample in 500 μL of DMSO-d6. The spectrum of RM-5071 was acquired with a proprietary NMR method ( ¹ H) using a 400 MHz instrument with D1 = 30 s, allowing the integrals of the peaks at 8.5 and 2.8 ppm to be quantified. .

1.2.4.3. Results

FIG. 17 contains the ¹ H NMR spectrum of the RM-5071 molecule and FIGS. 18 and 19 contain magnifications of various regions of the spectrum to assess the details of the peak pattern. A chemical shift (ppm) provides information of the chemical environment surrounding a particular hydrogen atom. The integration of the area under the peak provides information on how many protons are sensing the same chemical environment, whereas the peak splitting pattern provides information on nearby protons within the molecule.

The spectral peaks assigned based on the proposed salt molecular structure for RM-5071 containing a total of 14 protons are below.

Peak assignments indicate the presence of both expected counterions, ethanolammonium and tizoxanide alkoxide (desacetyl-NTZA). The singlet peak located at the maximum chemical shift of 17.701 ppm is assigned to the proton (H-13) within the amide group in the tizoxanide alkoxide adjacent to the negatively charged atom. The next singlet peak located at 8.503 ppm belongs to the hydrogen atom (H-14) in the thiazole ring of desacetyl-NTZA. The doublet doublet in the range 7.8-7.9 ppm is assigned to the proton of the aromatic proton designated H-9, which is the proton closest to the oxygen atom. The peak located at 7.673 ppm is assigned to three equivalent 'exchangeable' H atoms (H-1,2,3) adjacent to the nitrogen in the ethanolammonium ion. This is because the integral of the signal is 2.6, corresponding to about 3 protons, a "wide" singlet. All features are consistent with what is expected for labile amine protons. A complex splitting pattern located at about 7.3 ppm is assigned to the aromatic hydrogen located at position 10 (H-10). At first glance it resembles a triplet in which each peak is further split and located at a larger shift, to the hydrogen closer to the oxygen atom of the phenol with two unequal neighboring hydrogens. Because they match. A complex splitting pattern of multiplets located at about 6.8 ppm is summarized as signals from two hydrogens assigned to hydrogens 11 and 12. The singlet located at 5.129 ppm is assigned to the alcohol proton of ethanolammonium. The triplets located at 3.5 and 2.8 ppm are assigned to the aliphatic hydrogen H-6,7 near oxygen and H-4,5 near nitrogen in the ethanolammonium ion, respectively.

Integrals from ¹ HNMR spectra may not be precise enough to allow rigorous quantification. However, the integral data of the NMR peaks can be used to estimate the molar ratio between the ethanolammonium ion and the tizoxanide alkoxide ion in the RM-5071 salt. For example, set the area of the peak located at 8.50 ppm (H-14 of the thiazole ring) to 1.00 (reference). The integrals of the regions observed for the peaks located at about 2.8 ppm and 2.3 ppm (aliphatic H of ethanolamine) are then 1.956 and 1.952, respectively. This is consistent with the expected molar ratio between these two hydrogens given a 1:1 molar ratio between the ions.

1.2.5. Mass spectrum

Electrospray ionization (ESI) is one type of ionization technique in which analyte molecules are brought into the gas phase by forming charged droplets and then subjected to mass spectrometry (MS). These charged droplets undergo a process of desolvation when introduced into a vacuum that avoids fragmentation of molecular ions. ESI can be useful for generating ions from large organic molecules. This is because ESI overcomes the tendency of these molecules to fragment when ionized. Information obtained from ESI-MS spectra is useful for characterization purposes. This is because the molecular weight of the complete molecule can be derived from the mass spectrum obtained. Positive ion mass spectra of organic molecules typically consist of protonated species ([M+H] ⁺ , [M+2H] ^{2+ ,} etc.) and adducts of sodium, potassium, or other cations ( [M+Na] ⁺ , [M+K] ^+, etc.). Negative ion mass spectra typically consist of deprotonated species ([MH] ⁻ , [M-2H] ^{2− ,} etc.).

1.2.5.1. Materials and Equipment

The instrument used for direct injection electrospray ionization mass spectrometry (ESI-MS) at MCC is the research instrument Xevo G2-S QToF (quadrupole-time of flight) equipped with Mass Lynx data acquisition software. The instrument used for the HPLC-DAD-ESI-MS experiments in Landen (Belgium) was an Agilent 1100 HPLC with diode array detection (DAD), electrospray ionization (ESI) and Open Lab data acquisition software. InfinityLab MSD G6100 quadrupole mass spectrometry (MS) detection is done. The stream from the HPLC is split 2/3 to DAD and 1/3 to ESI-MS.

1.2.5.2. Procedure

A sample preparation of approximately 6 mg of RM-5071 was weighed. The RM-5071 sample was transferred to a 10 mL volumetric flask and filled to the mark with dimethylsulfoxide (DMSO) for positive-ESI and methanol for negative ESI. After further dilution of both samples, they were injected directly into the system. The instrument settings and instrument specifications are detailed in Table 16.

The Belgian procedure was to analyze samples of RM-5071 at concentrations between 0.61 mg/mL in dimethylformamide (DMF). This mixture was injected into an Agilent InfinityLab MSD G6100 with the following chromatographic conditions:

Absorbance chromatograms (DAD) and total ion chromatograms (MD) were generated as a function of time. Mass spectra of the tizoxanide alkoxide peak identified as the reference in the absorbance chromatogram were obtained in positive and negative modes.

1.2.5.3. Results

A summary of the dominant signals of ion m/z (ratio of mass to charge) for positive and negative ionization ESI-MS is available in Error! Reference source not found 18. error! Reference source not found contains a positive ESI-MS spectrum of RM-5071 dissolved in DMSO. Negative ion ESI-MS spectrum of RM-5071 in methanol is also in error! Reference source not found, where a fundamental peak located at m/z 264 was observed, corresponding to the [MH] ⁻¹ ion of the tizoxanide alkoxide.

Total ion chromatograms were obtained from experiments performed in Belgium after chromatographic separation. An electrospray ionization mass spectrum of the tizoxanide alkoxide peak from the total ion chromatogram is shown in FIG. The molecular ions observed by both positive and negative ESI-MS [M+1]+1 are m/z 266.10 and [M-1]-1 m/z 264.10, respectively, and these peaks represent tizoxanide It agrees with the expected mass of negative molecular ions of alkoxides.

1.2.6. FT-IR

Fourier transform infrared spectroscopy (FTIR) is a potentially valuable analytical technique for the characterization of organic molecules. FTIR records the absorption of infrared energy by vibrational modes related to stretching and bending of specific bonds in molecules. The resulting spectrum is a combination of the separate absorption properties of bonds and functional groups within the molecule. The FTIR spectrum thus becomes a molecular "fingerprint" for identification or comparison against known spectra. In the case of RM-5071 the spectrum can be compared with that of desacetyl-NTZA.

1.2.6.1. Materials and Equipment

The instrument used at MCC to perform the FT-IR analysis of RM-5071 and desacetyl-NTZA is a Thermo iS50 spectrometer equipped with a Continuum IR microscope. The FTIR instrument used for the FTIR analysis in Landen (Belgium) is a Shimadzu IRafinity fitted with an attenuated total reflectance accessory.

1.2.6.2. Procedure

A spatula was used to separate the sample. Once separated, the RM-5071 and desacetyl NTZA samples were mounted (individually) in two diamond microscopy cells and compressed to obtain thin films. The microscopy film was mounted on the stage of a Continuum IR microscope.

1.2.6.3. Results

FTIR techniques are commonly used to obtain a special "fingerprint" of a sample for identification or comparison against spectra of known compounds or compounds from computer data searches. Error in FTIR spectra of RM-5071 and desacetyl-NTZA! The reference source is not found. A summary of the functional groups identified for both samples is shown in Table 19. error! Reference source not found includes FTIR spectra obtained in Landen (Belgium).

A glance at the overlay of the FTIR spectra of RM-5071 and desacetyl-NTZA reveals similarities and significant differences that can be analyzed to obtain important information about the RM-5071 salt. The spectral differences are most likely due to the presence of ethanolammonium ions in RM-5071 and thus the different chemical environment surrounding the tizoxanide alkoxide counterion. Both spectra show a broad peak absorption in the region of 3000 cm ⁻¹ characteristic of the OH and NH stretching modes typically common to both molecules. The spectrum of desacetyl-NTZA shows a sharp strong peak at ~3250 cm ^-1 and a sharp but less intense peak at ~3100 cm ^-1 , which are absent in the spectrum of RM-5071. . In addition, there is a sharp and intense band at about 1670 cm ^-1 , which is characteristic of the carbonyl stretching mode typically present in desacetyl-NTZA spectra, but which is absent in the RM-5071 spectrum. completely disappeared. The disappearance of the amide carbonyl peak around 1672 cm ^-1 and the peaks at 3100 and 3250 cm ^-1 (presumably NH or OH signals) indicate the phenolate-ammonium-amide intramolecular coordination complex shown in Fig. 23. strongly contributes to the resonance.

The 'fingerprint' region of the spectrum below about 1500 cm ^-1 is expected to be attributed to the functional group contribution of the ethanolammonium ion and is different between both molecules. The disappearance of the amide carbonyl peak was also confirmed by the results of FT-IR experiments in Belgium. FT-IR analysis was also performed as a powder using an attenuated total reflectance (ATR) accessory. The results also showed clear differences in the carbonyl and NH stretching peaks between RM-5071 and desacetyl-NTZA. In addition, the analyzed 1:1 mixture of desacetyl-NTZA and ethanolamine showed a carbonyl peak at 1672 cm ⁻¹ that was different from the spectrum of RM-5071, thus this peak disappeared only in the salt and individual molecules It is confirmed that it does not disappear in a mixture of This favors the formation of salt complexes.

1.2.7. XRDs

X-ray diffraction is based on constructive interference between monochromatic X-rays and a crystalline sample. When the condition of Bragg's law (nλ=2d sin θ) is satisfied, constructive interference (and diffracted X-rays) occurs due to the interaction of the incident radiation with the sample. This law relates the wavelength of an electromagnetic wave to the diffraction angle and lattice spacing within a crystalline sample. X-ray crystallography is a tool used to identify the atomic and molecular structure of crystals, within which crystal atoms diffract a beam of incident X-rays into many specific directions.

1.2.7.1. Materials and Equipment

The instrument used by MCC to perform the analysis is the Rigaku SmartLab X-ray diffractometer.

1.2.7.2. Procedure

XRD analysis was performed on a Rigaku SmartLab X-ray diffractometer system equipped with a sealed copper cathode tube, Cu-K beta filter, and D/teX Ultra detector.

A temperature test was performed to determine if any physical and crystallinity changes occurred in sample RM-5071 when heated. To see if any changes occur, two sets of experiments were performed as follows:

Experiment A - Two petri dishes labeled "Fast" and "Slow". Approximately two spatulas of sample RM-5071 were added to each Petri dish. The furnace was then turned on and the temperature set to approximately 160-165°C. After reaching this temperature, the petri dish was placed in an oven for 15 minutes. When the time was up, the furnace was switched off and the 'fast' samples were removed and left in the hood to reach room temperature (RT). The "slow" samples were left in the furnace to reach RT more slowly. "Slow" samples were removed from the furnace when RT was reached. Samples were analyzed by XRD.

Experiment B - Followed the above procedure with a temperature range of approximately 110-120°C. After reaching that temperature, the Petri dish was placed in an oven for 10 minutes. Removal of samples from the furnace was as previously described. The samples were then analyzed by XRD.

1.2.7.3. Results

By measuring the angles and intensities of these diffracted beams produced by X-ray diffraction, a three-dimensional image of the density of electrons in the crystal can be produced. The RM-5071 sample showed a discernible XRD pattern, with diffraction signals between 8 and 37° 2θ angles. This suggests that this material exhibits a crystalline morphology. The desacetyl-NTZA sample showed a discernible XRD pattern with diffraction signals between 6 and 44° 2θ angles. This suggests that this material exhibits a crystalline morphology. RM-5071 and desacetyl-NTZA showed different XRD patterns. This suggests that they exhibit different crystal morphologies (Error! Reference source not found AB).

A temperature test was performed to determine if any physical and crystallinity changes occurred in RM-5071 upon heating. The temperatures for these heating experiments were chosen based on the TGA transition identified in Figure 11, experiment A heated to 160-165°C and experiment B heated to 110-120°C. The results of this heating experiment are summarized in Table 20. The results show that in experiment A the appearance of the sample changed from bright yellow to black powder with beige areas. The XRD pattern of the beige part resembles that of desacetyl-NTZA. The XRD diffractogram of the black powder resembled neither that of desacetyl-NTZA nor that of RM-5071, which was characteristic of an amorphous material. The results of experiment B show that the material does not change significantly at 110-120°C, as evidenced by the lack of physical change in the powder and XRD patterns before and after heating.

2. Conclusion

This report discusses the accumulated chemical and physical characterization data for RM-5071. In addition, the chemical and physical properties of RM-5071 were compared with those of its precursor desacetyl-NTZA.

RM-5071 is a yellow powder with an average particle size of approximately 11 μm, as revealed by laser diffraction analysis and confirmed by scanning electron microscope images. RM-5071 forms agglomerates and can be dispersed by mechanical action, sonication, or pneumatic pressure in a venturi-type dispersing device. The melting point is in the range of 146-148°C. Thermal properties of the material, investigated by thermogravimetric analysis and differential scanning calorimetry, show that the solid is stable up to 163°C, where it undergoes an irreversible transition. This temperature is below the first transition temperature of desacetyl-NTZA. XRD reflects that RM-5071 has a more distinct crystal structure than desacetyl-NTZA.

RM-5071 is, according to the USP definition, DMF, DMSO, ACN: very soluble in water, but not even slightly soluble in water. RM-5071 is soluble in methanol, ethanol, and isopropanol (<0.033 mg/mL) and very soluble in binary mixtures of water:ethanol or isopropanol 25:75 and 50:50. The pKa of the phenolic proton of RM-5071 is determined to be 8.8 by acid-base titration. Characterization by UV-Vis showed peaks in the visible region ranging from 409 to 431 nm for all solvents. Electrospray mass spectrometry results show evidence for the presence of desacetyl-NTZA in solution, most easily detected in both negative and positive modes after chromatography of the RM-5071 sample. , the expected mass/charge ratio is confirmed. The FTIR data may suggest the existence of a proposed equilibrium between tautomers, the reason being that in the IR spectrum of RM-5071, the spectrum of pure desacetyl-NTZA , and the disappearance of the characteristic carbonyl band compared to the spectrum of a 1:1 mixture of desacetyl-NTZA and ethanolamine. FTIR spectra can be used to distinguish RM-5071 from its precursor.

Although reference has been made thus far to certain preferred embodiments, it will be understood that the invention is not so limited. Those skilled in the art will realize that various modifications to the disclosed embodiments are possible and that such modifications are intended to be within the scope of the invention.

All publications, patent applications, and patents cited herein are hereby incorporated by reference in their entirety.

Claims (46)

formula:
Amine-containing salts of compounds with (where R is _NO2 or halogen). 2. The amine-containing salt of claim 1, wherein R is _NO2 . 2. The amine-containing salt of claim 1, wherein R is Cl. 4. The amine-containing salt of any one of claims 1-3, which is an alkylamine salt, an alkoxyamine salt, or a cycloalkylamine salt. 5. The amine-containing salt of any one of claims 1-4, which is the ethanolamine salt of said compound. 5. The amine-containing salt of any one of claims 1-4, which is the morpholine salt of said compound. 5. The amine-containing salt of any one of claims 1-4, which is the propanolamine salt of said compound. 5. The amine-containing salt of any one of claims 1-4, which is the N-methylpiperazine salt of said compound. 9. The amine-containing salt of any one of claims 1-8, which is a crystalline salt. A batch of amine-containing salt according to any one of claims 1 to 9 having a purity of at least 90%. 11. The batch of claim 10, containing at least 100 g of amine-containing salt. A pharmaceutical composition comprising the amine-containing salt of any one of claims 1-9 and a pharmaceutically acceptable excipient. 13. The pharmaceutical composition of claim 12, which is an oral pharmaceutical composition. 14. The pharmaceutical composition of claim 12 or 13, which, when administered to a mammal, provides maximum concentration of said compound in the plasma of the mammal in less than 1 hour. 14. The pharmaceutical composition of claim 12 or 13, wherein R is _NO2 and, when administered to a mammal, provides a maximum concentration of said compound in the plasma of said mammal sooner than a pharmaceutical composition comprising nitazoxanide. wherein R is _NO2 and provides an AUC _0-12h concentration of said compound in the plasma of said mammal that is equal to or greater than the AUC _0-12h concentration of a pharmaceutical composition comprising nitazoxanide when administered to said mammal. 12 or 13 pharmaceutical compositions. wherein R is _NO2 and, when administered to a mammal, the AUC _0-12h concentration of said compound and the glucorono form of said compound in the plasma of said mammal is greater than or equal to the AUC _0-12h concentration of a pharmaceutical composition comprising nitazoxanide 14. The pharmaceutical composition of claim 12 or 13, provided in A pharmaceutical composition comprising a salt of tizoxanide and a pharmaceutically acceptable excipient that provides a maximum concentration of tizoxanide in the plasma of a mammal in less than 1 hour when the composition is administered to the mammal. , pharmaceutical compositions. A pharmaceutical composition comprising a salt of tizoxanide and a pharmaceutically acceptable excipient, wherein when the composition is administered to a mammal, it is formed in the plasma of said mammal faster than a pharmaceutical composition comprising nitazoxanide A pharmaceutical composition that provides a maximum concentration of tizoxanide. A pharmaceutical composition comprising a salt of tizoxanide and a pharmaceutically acceptable excipient, wherein, when the composition is administered to a mammal, tizoxanide and glucoronotizoxanide are produced in the plasma of said mammal. A pharmaceutical composition that provides an AUC _0-12h concentration equal to or greater than the AUC _0-12h concentration of a pharmaceutical composition comprising nitazoxanide. The pharmaceutical composition according to any one of claims 18-20, which is an oral composition. The pharmaceutical composition of any one of claims 18-21, wherein said salt is the ethanolamine salt of tizoxanide. A method for producing an amine-containing salt of a thiazolide compound, comprising:
formula:
wherein R is NO ₂ or Cl with an amine-containing compound to form an amine-containing salt of the thiazolide compound. 24. The method of claim 23, wherein R is _NO2 . 24. The method of claim 23, wherein R is Cl. 26. The method of any one of claims 23-25, wherein said amine-containing compound is a liquid amine-containing compound. 27. The method of claim 26, wherein said amine-containing compound is an alkylamine, alkoxyamine, or cycloalkylamine. 28. The method of Claim 27, wherein said amine-containing compound is ethanolamine and said amine-containing salt is an ethanolamine salt of a thiazolide compound. 28. The method of Claim 27, wherein said amine-containing compound is morpholine and said amine-containing salt is a morpholine salt of said compound. 28. The method of claim 27, wherein said amine-containing compound is propanolamine and said amine-containing salt is a propanolamine salt of said compound. 28. The method of claim 27, wherein said amine-containing compound is N-methylpiperazine and said amine-containing salt is the N-methylpiperazine salt of said compound. 32. The method of any one of claims 23-31, wherein the amine-containing salt produced has a purity of at least 90%. 33. The method of claim 32, wherein said amine-containing salt is produced in an amount of at least 100 g. 34. The method of any one of claims 23-33, wherein said reaction is carried out in an alcoholic solution. 35. The method of Claim 34, further comprising crystallizing said amine-containing salt formed. formula:
Transform the prodrug thiazolide compound of the formula:
36. The method of any one of claims 23-35, further comprising producing a thiazolide compound of 37. The method of Claim 36, comprising heating said prodrug thiazolide compound. Ethanolamine salt of tizoxanide. 39. The salt of claim 38, having a particle size of about 4 microns to about 40 microns. 40. The salt of claim 38 or 39, having a melting temperature of about 146°C to about 148°C. 41. The salt of any one of claims 38-40 in crystalline form. 42. The salt of any one of claims 38-41, having a differential scanning calorimetry (DSC) curve as in Figure 12A. It has an X-ray powder diffractogram obtained with a diffractometer using Cu-Kβ rays with a wavelength of 1.39222 Å, and the diffractograms are 8.5°±0.2°, 11.2°±0.2°, 16.8°±0.2°, and 19.5°. 43. The salt of any one of claims 38-42, having peaks at °±0.2°, 20.9°±0.2°, 25.6°±0.2°, 27.0°±0.2°, and 36.1°±0.2° two-theta. 43. The salt of any one of claims 38-42, having an X-ray powder diffractogram as in Figure 25A determined with a diffractometer using Cu-Kβ radiation of wavelength 1.39222 Å. A batch containing at least 0.8 kg of the ethanolamine salt of any one of claims 38-44. 46. The batch of claim 45, wherein the molar ratio between ethanolamine and tizoxanide contained therein is between 0.9 and 1.00.